The Old Main Line Subdivision is a railroad line owned and operated by CSX Transportation in the U.S. state of Maryland. The line runs from Relay (outside Baltimore) west to Point of Rocks, and was once the main line of the Baltimore and Ohio Railroad, one of the oldest rail lines in the United States. At its east end, it has junctions with the Capital Subdivision and the Baltimore Terminal Subdivision; its west end has a junction with the Metropolitan Subdivision.

The initial route of the Baltimore and Ohio Railroad (B&O) followed the Patapsco River valley west out of Baltimore, with the first section (to what is now Ellicott City, Maryland) opening for service in 1830. The line left the valley to cross Parr's Ridge, which, after an abortive attempt to use a system of inclined planes, was crossed via a more round-about routing through Mount Airy. It continued west to Harper's Ferry, West Virginia, passing south of Frederick on the way. This line was the only route west out of Baltimore until the Metropolitan Branch was constructed from Washington, DC to Point of Rocks in the 1870s. The section of the original route between Relay (where the Washington Branch began) and Point of Rocks became known as the "Old Main Line" (OML), alluding to its subsidiary status, and continues to be known as the Old Main Line Subdivision in CSX timetables.

With railroad technology in its infancy, the engineers of the B&O made many design decisions that quickly proved to be mistaken. For instance, the route was laid out to minimize grades at the expense of curvature; over the next century, however, to eliminate and bypass the sharp curves that resulted from this decision, bridges and tunnels were constructed. The planes over Parr's Ridge also resulted from this same thinking, and subsequently gained the distinction of becoming one of the first railroad main line right-of-way abandonments in history.

Initially, a system of granite stringers and strap rail was preferred, although time, expense, and difficulty in obtaining sufficient granite led to the substitution of wooden ties and heavier "T-rails" for much of the route, beginning in the 1840s. In the 1850s, when Benjamin Henry Latrobe, II was chief operating engineer, the need to address these deficiencies became acute, and a variety of improvements were made, subject to the railroad's limited resources at the time. All of the granite stringers and strap rail were replaced, and certain realignments were made. Among these was the "Elysville cutoff," where a pair of bridges were constructed to bypass a sharp curve on the south side of the river. In making these improvements, older structures were simply abandoned. The granite stringers of the original roadbed were simply left in place and buried.

B&O built its first tunnel in 1850 at Henryton. The Henryton Tunnel was widened for double track in 1865, after the Civil War.

In 1868, a freak storm flooded the Patapsco and severely damaged the railroad, as well as washing away many of its customers. Most of the railroad was rebuilt, but with many alterations to the surviving structures. For instance, all but one arch of the Patterson Viaduct at Ilchester were washed away; the railroad retained the remaining arch to use as an abutment for the Bollman truss bridge which replaced the viaduct.

The first station on the line was built in Ellicott City in 1830, Over the years this station was modified and enlarged, and it survives to this day. The next station erected was a freight depot in Frederick, built 1831. Another simple station was built in Mt. Airy, which also survives.

In the 1870s and 1880s, the railroad undertook a program of station building. Most of these were designed by E. Francis Baldwin and several towns on the Old Main Line received such stations, erected in either wood or brick. The most famous of these, Point of Rocks, still stands and is still in use in the wye between the OML and the Metropolitan Subdivision. Other stations were built at Sykesville, Ilchester, and Woodstock, though not all survive.