In its initial duty, it provided security near the capitol and in January, it became part of the provisional brigade of Casey's division.

During McClellan's building of the AoP, on March 13, it joined the 1st brigade, 1st division, II Corps, and went to the Peninsula with the army.

The regiment was present during the siege of Yorktown, but received its first real test at Fair Oaks, where it behaved with great steadiness under a fire which killed or wounded 173 of its members.

[8] At Fredericksburg, in the famous assault of Hancock's division on Marye's heights, the loss of the regiment was 72 in killed and wounded and immediately afterward it went into camp near Falmouth.

During the winter of 1863-64 a sufficient number of the regiment reenlisted to secure its continuance in the field as a veteran organization, but after the original members not reenlisted were mustered out in the autumn of 1864 it was necessary to consolidate it into a battalion of six companies, for which new people were recruited, A, B, D, E, G and H.[9] It served through the Wilderness campaign, throughout the siege of Petersburg and in the pursuit of Lee's Army to Appomattox, losing 16 in killed and wounded at Farmville.

[10] The division in which it served saw the "hardest service and suffered the most heavy losses of any in the army and the 64th was one of the finest fighting regiments in the war.

By the end of the first full year of hard campaigning, the regimented reported possession of 344 Enfield P1853s, 424 Mle 1842, and 62 Prussian Potsdam smoothbore percussion muskets (.71 caliber).