U.S. Route 27 (US 27) is a north–south United States highway in the southern and midwestern United States. The southern terminus is at US 1 in Miami, Florida. The northern terminus is at Interstate 69 (I-69) in Fort Wayne, Indiana. From Miami it goes up the center of Florida, then west to Tallahassee, Florida, and north through such cities and towns as Columbus, Georgia; Rome, Georgia; Chattanooga, Tennessee; Lexington, Kentucky; Cincinnati, Ohio; Oxford, Ohio; Richmond, Indiana and Fort Wayne, Indiana. It once extended north through Lansing, Michigan, to Cheboygan, Mackinaw City, and for about 3 years as far as St. Ignace.

US 27 appeared in 1926, replacing what had been the western route of the Dixie Highway in many places.

In Florida, US 27 has been designated the Claude Pepper Memorial Highway by the Florida State Legislature. It was named after long-time Florida US Senator and congressman Claude Pepper. Nearly the entire length of US 27 in Florida is a divided highway.

US 27 begins as North 36th Street in Midtown Miami, heading west from US 1 for 4.4 miles (7.1 km) before turning northwest to pass under the western terminus of the Airport Expressway (State Road 112, SR 112). It then proceeds northwest for five miles (8 km) as South Okeechobee Road, parallel to the Miami Canal, forming the southwest boundary of the city of Hialeah. After an interchange with the Palmetto Expressway (SR 826), it continues northwest as North Okeechobee Road for five miles (8 km) before an interchange with the Homestead Extension of the Florida Turnpike (SR 821). After another four miles (6 km), the highway curves to the north and, after passing the northern terminus of Krome Avenue (SR 997), crosses into Broward County.

In Broward County, the highway passes protected wetlands and heavy duty power lines on the west and the outer reaches of the suburban communities of Pembroke Pines and Weston on the east. US 27 then reaches an interchange with I-75 and Alligator Alley before curving to the northwest toward South Bay and Lake Okeechobee.

The highway skirts the southwestern shore of Lake Okeechobee and then heads west at Clewiston, before making a sharp turn to the north towards Moore Haven. The road then proceeds in a northerly direction toward the central Florida communities of Lake Placid, Sebring, Avon Park, and Lake Wales.

Widening of US 27 to a six-lane highway continues in Polk County. The following sections have been completed and are open to six lanes of traffic:

North of I-4, US 27 contains un-numbered interchanges with US 192 and then County Road 474 (CR 474) in Citrus Ridge, SR 50 in Clermont, and SR 19 south of Howey-in-the-Hills, which also includes a southbound interchange with Florida's Turnpike. The northbound Turnpike interchange can be found further northwest. US 441 later joins US 27 in Leesburg and US 301 in Belleview, only for the road to break away from both in Ocala.

US 27 resumes its status as its own route until it reaches Williston and joins US 41. This concurrency continues northward until US 41 reaches High Springs, and joins US 441. US 27 heads west along the unsigned SR 20 towards Perry and joins US 19 until US 19 breaks away in Capps, but not before resuming a westward direction. In Tallahassee, the road becomes Apalachee Parkway is a major east–west thoroughfare. Constructed in 1957, the Apalachee Parkway starts at Monroe Street in front of the Florida State Capitol building. It has a short expressway section just east of the capitol, then is a busy four-lane surface boulevard with service roads for the next few miles, passing the Governor's Square Mall and many state office buildings. Past Tallahassee, US 27 finally resumes its northwesterly direction. The highway goes through Havana before entering Georgia.

In Georgia, US 27 has been designated the Martha Berry Highway by the Georgia State Legislature. It was named after Martha Berry, founder of Berry College in Rome. US 27 is a designated Governor's Road Improvement Program (GRIP) developmental highway corridor which will eventually be widened to four lanes (mostly divided) from the Florida state line to the Tennessee state line. All of US 27 in Georgia is concurrent with Georgia State Route 1.

In Chattanooga, Tennessee, a portion of US 27 was once signed as I-124. Though the designation still exists, it is no longer signed as such. In and around the Chattanooga area, US 27 is sometimes referred to as Corridor J, the designation of a road in the Appalachian Development Highway System between Chattanooga and London, Kentucky intended to follow the route of US 27.

From Chattanooga, the highway passes through Soddy-Daisy and the small communities of Sale Creek and Graysville before reaching Dayton, the site of the infamous Scopes Monkey Trial. It then passes through Evansville before arriving at Spring City.

From here, US 27 enters Roane County, running concurrent with US 70 going through the city of Rockwood. After US 70 splits to the east, US 27 runs concurrent with State Route 61 (SR 61) through Harriman, where it is crossed by I-40. During this stretch, it forms part of the Harvey H. Hannah Memorial Highway, and is signed as such.

In Morgan County, the highway passes through Wartburg and Sunbright. It passes through Robbins and Helenwood before reaching Oneida in Scott County.

Winfield is the last sizable town that US 27 passes through in Tennessee before it reaches the community of Isham on the Kentucky border.

US 27 enters Kentucky near Whitley City. The first major town it goes through is Somerset, followed by Nicholasville, and Lexington, where it begins a concurrency with US 68 that continues to Paris. US 27 continues north from there through Cynthiana, Falmouth, Alexandria, Highland Heights, Fort Thomas, and Newport before crossing into Ohio at Cincinnati via Taylor-Southgate Bridge. From its junction with Interstate 471 in Highland Heights through Campbell County, it is a four lane highway with a center turn lane for both directions of traffic. Near the "welcome to Alexandria" sign at KY 536, US 27 becomes a divided four lane highway with a grass median. It is currently being constructed as such for the rest of its length from 536 through southern Campbell county. Currently, US 27 is under-going a widening project near Garrard CR 1355 to HWY 34. The road will become a four lane into Lexington.

US 27 crosses into Cincinnati via the Taylor–Southgate Bridge. The road continues Northwest from Oxford until reaching the Indiana border at College Corner.

US 27 enters the southeastern part of Indiana south of the town of Liberty. At Liberty, US 27 turns north, paralleling the Ohio state line. It intersects I-70 at Richmond, Indiana, and continues north to Decatur. US 33 joins US 27 just south of Decatur and the two routes share a northwesterly alignment on a divided surface highway from Decatur to I-469 south of Fort Wayne. US 33 turns west and follows I-469, while US 27 continues north into downtown Fort Wayne as Clinton Street. North of downtown, US 27 follows Lima Road to its terminus at I-69/US 30 (Lima Road continues north from this point as SR 3).

Originally, the southern terminus of US 27 was in Cincinnati, Ohio. In 1928, the route was extended south to Chattanooga, Tennessee, then to Tallahassee, Florida in 1934. In 1949, it was extended to its current terminus in Miami. At its northern end, US 27 originally terminated at Cheboygan, Michigan. In 1937, the route was extended concurrently with US 23 to Mackinaw City, where it ended at the Michigan State Auto Ferry Dock along with US 23 and US 31. After the completion of the Mackinac Bridge in 1957, US 27 was extended across the bridge to the current intersection of I-75 and US 2 near St. Ignace. In 1961, what became I-75 was completed, enabling the roadway to be designated as I-75. At the same time, US 27 was truncated to an interchange with I-75 six miles (10 km) south of Grayling. In 2002, the northern terminus was moved to its current location.

From Grayling to I-69 near Lansing, the road is now designated US 127. US 27 signs were removed from I-69 between Lansing and Fort Wayne in 2001. Officially, the current northern terminus is at the junction of I-69, US 30, and SR 3, but heading north on I-69 one will still see US 27 shields on some of the older overhead signs heading to the I-469 interchange on the northern edge of Fort Wayne.

On March 9, 2007, legislation was introduced in the Indiana House of Representatives to designate US 27 as historic highway. Richmond, one of the cities US 27 passes through, would have two historic highways passing through it. The original National Road (US 40) runs through Richmond.

US 27 was one of the highways in Michigan to carry the TEMP I-75 designation when I-75 was being constructed along then M-76.

The at-grade routing of US 27 in Michigan has been largely superseded by I-75, which follows a roughly similar route and in some places actually replaced the older highway. From Lansing north to I-75, the former US 27 is now US 127; from Lansing south to Fort Wayne, US 27 was replaced by I-69. In many places the route markers and the highway itself serve to connect local communities bypassed by the Interstate Highway.