CSX Transportation was formed on July 1, 1986 as a renaming of the Seaboard System Railroad and Chessie System, Inc. into one entity. The originator of the Seaboard System was the former Seaboard Air Line Railroad, which previously merged Atlantic Coast Line Railroad, and later Louisville and Nashville Railroad, as well as several smaller subsidiaries. On August 31, 1987 the Chesapeake and Ohio Railway, which had absorbed the Baltimore and Ohio Railroad on April 30 of that year, merged into CSX. The merger had been started in 1980 with the merger of Chessie System and Seaboard Coast Line Industries to form the CSX Corporation.

On June 23, 1997, CSX Transportation and Norfolk Southern filed a joint application with the Surface Transportation Board for authority to purchase, divide and operate the assets of the 11,000-mile Consolidated Rail Corporation (Conrail), which had been created in 1976 by bringing together several ailing Northeastern railway systems into a government-owned corporation. On June 6, 1998, the STB approved the CSX-Norfolk Southern application and set August 22, 1998, as the effective date of its decision. CSX acquired 42% of Conrail's assets, and Norfolk Southern received the remaining 58%.

As a result of the transaction, CSX's rail operations grew to include some 3,800 miles of the Conrail system (predominantly lines that had belonged to the former New York Central Railroad). CSX began operating its trains on its portion of the Conrail network on June 1, 1999. CSX now serves much of the eastern U.S., with a few routes into nearby Canadian cities.