The nucleus of the regiment was a battalion of six companies raised in September 1861 to garrison Fort Warren, the largest fortification in Boston harbor.

The unit was transferred to the battle front following Abraham Lincoln's urgent call for troops in response to the Confederate advance on Washington during Jackson's Valley Campaign in May 1862.

In July 1862, the 32nd was assigned to the Army of the Potomac and was shipped to Fortress Monroe to join its new command at the close of the unsuccessful Peninsular Campaign.

In the Overland Campaign, during the Battle of Spotsylvania Court House, the unit was heavily engaged and suffered 54 percent casualties—its worst casualties of the war.

[2] The composition of the 32nd Massachusetts was unusual in that it was continually replenished, during the latter months of the war, by men from disbanded regiments who had reenlisted for a second term of service.