Herpesviruses are significant pathogens of captive-reared and free-ranging chelonians worldwide. Lesions associated with chelonian herpesvirus infection are species dependent and include stomatitis in tortoises, hepatic necrosis in pond turtles, and fibropapillomas in sea turtles, among other conditions. Herpesviruses also have been detected in several free-ranging freshwater turtle species with no clinical signs of illness at a prevalence from 2 to 56%. The alligator snapping turtle (Macrochelys temminckii) is a freshwater turtle species endemic to the United States that has experienced declines throughout its range. Reintroduction of this species is currently underway in several states. As part of a health surveillance program, in concert with reintroduction efforts, we investigated the presence of herpesvirus in captive and free-ranging alligator snapping turtles. Using conventional consensus polymerase chain reaction, we tested combined oral/cloacal swab DNA samples (n = 197) from head-started alligator snapping turtles prerelease and postrelease in southern Illinois (n = 153), prerelease samples from adult turtles confiscated from Florida (n = 18), and prerelease samples from captive-reared individuals from northern Louisiana (n = 26). Herpesvirus DNA was not detected in any sample. Possible explanations for these results include lack of exposure, latency in tissues not sampled, and viral quantities below the level of detection of the assay. Continued surveillance for this and other pathogens is helpful in characterizing potential disease risks from captive-reared reintroduction programs and will enhance future conservation efforts of this species.