Multifocal hyperemic nodules and plaques associated with the cloacal mucosa of juvenile alligators (Alligator mississippiensis) at a public aquarium were investigated. Grossly, pale pink to dark red multifocal, circular lesions of varying degrees of severity were identified on the cloacal and, in males, phallus mucosa. Cloacal mucosa biopsies were obtained from 2 of the alligators. These samples were examined histologically and by polymerase chain reaction (PCR) using consensus primers targeting a conserved region of the herpesvirus polymerase gene. Microscopically, the lesions were characterized as submucosal lymphoid follicles with hyperemia and hemorrhage. No inclusion bodies were observed. Minimal to no anisokaryosis was present, and no etiologic agents were identified. Through PCR, a band consistent in size with herpesvirus was observed. Tissues showing similar clinical, histopathologic, and PCR findings were collected from animals at an alligator farm several months later. Sequencing of the PCR amplicon resulted in a 180-base pair sequence that shared 85% sequence identity with tortoise herpesvirus-1.