Transported to Washington, D.C., the members of this regiment were initially attached to Division of the Potomac (Sickles' Brigade), and assigned to defend the city before participating in the Union Army's expedition to lower Maryland (September 15–October 2, 1861).

[5][6] Engaged later that summer in the Union's Northern Virginia Campaign, under the command of Major-General John Pope, and in operations at Fairfax Station and along the Orange and Alexandria Railroad that fall, the 72nd New York received roughly 200 replacement soldiers in December 1862, bringing the regiment's strength to just over 400.

It was then ordered into the Battle of Fredericksburg (December 12–15), where it remained in the field under arms and without tents for 50 hours before retreating with other Union Army troops.

[5][6] Encamped at Falmouth, Virginia through April 1863, the regiment participated in Major-General Ambrose Burnside's Mud March (January 1863) and in operations at Rappanhannock Bridge and Grove Church (February 5–7) before being ordered on to the Chancellorsville Campaign.

During this phase of duty, the 72nd New York lost nearly one-fourth of its members in the intense combat of the Battle of Chancellorsville on May 7, including the regiment's commanding officer, Colonel William O. Stevens, who died the next day after having been mortally wounded and captured by Confederate States Army troops.