U.S. Route 27 or U.S. Highway 27 (US 27) is a north–south United States Numbered Highway in the Southern and Midwestern U.S. 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 three years, even as far as St. Ignace. US 27 was first signed in 1926, replacing what had been the eastern route of the Dixie Highway in many states.

Lexington is the second-most-populous city in the Commonwealth of Kentucky, and the 60th-most populous city in the United States. It is the county seat of Fayette County. By land area, it is the country's 30th-largest city.