An Alternate U.S. 27 routing exists in Florida. It is signed north–south, but is a primarily east–west route, running between Perry and Williston. ALT US 27 is concurrent with US 19-98 from Perry to Chiefland, and also has the hiddenFDOT designation of State Road 55. Between Chiefland and Williston it is (hidden) SR 500. The entire length of the route is 93 miles (150 km).
Alternate U.S. 27 is one of the primary routes between northwest and central Florida, having been recently been four-laned along its entire route. With a signed speed limit of 65 mph (105 km/h), this gives it more capacity than the mostly-two-lane mainline route.
US 27 Bus. / SR 1 Bus. is a 3.473 miles (5.589 km)-long business branch of US 27 and SR 1 in Attapulgus, which leaves the main route of US 27 and SR 1 at mile 6.349, and heads northwest in the direction of downtown Attapulgus, angling west through the heart of the town, and then turning sharply north at the western edge of Attapulgus to meet US 27/SR 1 again at mile 9.225.
US 27 Bus. / SR 1 Bus. in Bainbridge continues north into Bainbridge at mile 18.627, where the main route of US 27 starts its concurrency going west with US 84 and SR 38 around town. US 27 Bus runs to its intersection with the US 84 Bus/SR 38 Bus branch originating east of Bainbridge, and runs west for a short distance, co-signed with these two routes. US 27 Bus then turns sharply north again at Broad Street, where it starts a concurrency with SR 97 and SR 309. The route splits from SR 97 and SR 309 after a short distance, and runs northwest to its terminus as it rejoins the main US 27 route out of Bainbridge, and is briefly co-signed with SR 253 during this portion. The entire route is in Bainbridge, Decatur County.
US 27 Bus. / SR 1 Bus. in Blakely is also named Main Street, and runs south-to-north through the center of town. The business route is also co-signed with SR 39 for a majority of its length, and is further co-signed with SR 62 from just south of to the eastern portion of Courthouse Square around the Early County Courthouse.
US 27 Bus. / SR 1 Bus. in Cuthbert is also named Blakely Street south of the Randolph County Courthouse square, and Court Street north of the square. The route runs south-to-north through the heart of town, crossing US 82 and SR 50 at the courthouse square.
State Route 1 Connector (SR 1 Connector) is an old alignment of US 27, but currently carries only a designation related to State Route 1, US 27's assigned state route number. It is a short south-to-north connecting route in Lumpkin between SR 27, also named Broad Street, which runs west-to-east through the heart of downtown, to US 27/SR 1 north of Lumpkin. SR 1 Connector is also called Chestnut Street; however, Chestnut Street extends south of SR 26, while the SR 1 Connector designation is only valid of the portion north of SR 27.
US 27 Bus. / SR 1 Bus. in Bremen swings through downtown Bremen from just north of I-20, from a southern terminus in Carroll County, while US 27 bypasses the town to the west. The route crosses US 78 and SR 8 in the heart of downtown.
State Route 1 Loop (SR 1 Loop) is not related to US 27, but to its concurrent state route number, State Route 1. It forms a partial loop around downtown Rome, which starts west of the US 27 main route on SR 20, runs first north, then west, and crosses the main route of US 27 in Mount Berry north of Rome. The loop then continues to run clockwise west and south to meet its southern terminus, again at SR 20 and its concurrency with US 411.