Companies operating former Atlantic Coast Line Railroad lines

The Georgia and Florida Railway (reporting mark GFRR) is a short line railroad operating in Georgia and Florida, and is a subsidiary of OmniTRAX. The railroad spans 297 miles (478 km) over numerous different rail lines, most of which radiate out of Albany, Georgia.

The Florida West Coast Railroad (reporting mark FWCR) was a 13-mile (21-kilometre) railroad owned by CSF Acquisition, Inc., which acquired it from CSX on December 13, 1987 as its first acquisition. It ran west from a CSX line at Newberry to Trenton.

The Florida Midland Railroad Company, Inc. (reporting mark FMID) is one of several short line railroads operated by Regional Rail, LLC in Florida (along with the Florida Central Railroad and the Florida Northern Railroad). The Florida Midland Railroad operates two former CSX Transportation railroad lines including their former Lake Wales Subdivision (running from West Lake Wales to Frostproof), and their former Bartow Subdivision (from Winter Haven to a point just northwest of Bartow). It once had a third line from Wildwood to Leesburg that is now mostly abandoned. Florida Midland Railroad began operating the lines in 1987 and uses locomotives branded for the Florida Central Railroad, its sister railroad.

The Florida Central Railroad Company (reporting mark FCEN) is one of several short line railroads run by Regional Rail, LLC. It runs from downtown Orlando northwest to Apopka and Tavares with a branch from Toronto to Ocoee and Winter Garden and branches from Tavares to Umatilla and Sorrento. The Florida Central connects with the Central Florida Rail Corridor in downtown Orlando, Florida and has trackage rights on the CFRC from there south to Taft Yard where they interchange with CSX Transportation. The railroad is based out of the Plymouth freight station.

SunRail (reporting mark CFRC) is a commuter rail system in the Greater Orlando, Florida, area. Services began on May 1, 2014. The system comprises 17 stations along a former CSX Transportation line connecting Volusia County and Osceola County through Downtown Orlando. The SunRail system is financed by the state and federal governments and the counties it serves. SunRail is Florida's second commuter rail system after South Florida's Tri-Rail.

Carolina Southern Railroad (reporting mark CALA) was located off of Main Street in Conway, South Carolina, and was one of the few remaining train depots/lines in South Carolina. The railroad was originally erected in late 1886 and the first train steamed into the Conway Depot in December 1887. R.J. Corman Railroad Group took over the operations in 2015.

The Seminole Gulf Railway (reporting mark SGLR) is a short line freight and passenger excursion railroad headquartered in Fort Myers, Florida, that operates two former CSX Transportation railroad lines in Southwest Florida. The company's Fort Myers Division, which was previously the southernmost segment of CSX's Fort Myers Subdivision, runs from Arcadia south to North Naples via Punta Gorda, Fort Myers, Estero, and Bonita Springs. The company's other line, the Sarasota Division, runs from Oneco south through Sarasota. Seminole Gulf acquired the lines in November 1987 and operates its own equipment. The company's first train departed Fort Myers on November 14, 1987.

The South Central Florida Express, Inc. (reporting mark SCXF) (originally known as the South Central Florida Railroad (reporting mark SCFE)) is a common carrier shortline railroad in southern Florida run by U.S. Sugar Corporation. Its trains operate from Sebring to Fort Pierce via Clewiston around the southern perimeter of Lake Okeechobee, and serves customers at 26 locations. With 171 miles (275 km) of track, the SCXF is the largest private agricultural railroad in the U.S.

The Norfolk Southern Railway (reporting mark NS) is a Class I freight railroad operating in the Eastern United States. Headquartered in Atlanta, the company was formed in 1982 with the merger of the Norfolk and Western Railway and Southern Railway. The company operates 19,420 route miles (31,250 km) in 22 eastern states, the District of Columbia, and has rights in Canada over the Albany to Montreal route of the Canadian Pacific Kansas City. Norfolk Southern Railway is the leading subsidiary of the Norfolk Southern Corporation.

CSX Transportation (reporting mark CSXT), known colloquially as simply CSX, is a Class I freight railroad company operating in the Eastern United States and the Canadian provinces of Ontario and Quebec. Operating about 21,000 route miles (34,000 km) of track, it is the leading subsidiary of CSX Corporation, a Fortune 500 company headquartered in Jacksonville, Florida.