U.S. Highway 27 (US 27) in Florida is a north–south United States Numbered Highway. It runs 496.352 miles (798.801 km) from the Miami metropolitan area northwest to the Tallahassee metropolitan area. Throughout the state, US 27 has been designated the Claude Pepper Memorial Highway by the Florida Legislature. It was named after long-time Florida statesperson Claude Pepper, who served in both the U.S. Senate and House of Representatives. The stretch running from Miami to South Bay was originally designated the Thomas E. Will Memorial Highway by the Florida Legislature in 1937 when that portion was known as State Road 26 (SR 26). Thomas E. Will, the founder of Okeelanta, had worked for almost 20 years to get the state to build a road from Miami to the area south of Lake Okeechobee. For most of its length in the state, US 27 is a divided highway.

State Road 500 is a state highway running through Florida as a mostly unsigned route under several U.S. Highways. From Chiefland to Williston it is U.S. Highway 27 Alternate. From Williston to Ocala, it is US 27. From Ocala to Holopaw, it is US 441. From Kissimmee to Indialantic it is US 192.