We studied the pathology, epidemiology, and clinical significance of genital herpesvirus infection in a zoo collection of bottlenose dolphins (Tursiops truncatus). Samples from the genital mucosa of male (n=21) and female (n=15) dolphins were tested by nested polymerase chain reaction (PCR) targeting the DNA polymerase of herpesvirus. Herpesvirus infection was significantly associated with the occurrence of mucosal plaques on penis (n=3) or vulva (n=4). Biopsies from a penile plaque showed epithelial hyperplasia by histology, contained herpesvirus-like particles by electron microscopy, and tested positive for herpesvirus by PCR. Herpesvirus was successfully cultivated from penile plaque samples and identified as a member of the Gammaherpesvirinae by DNA sequencing and phylogenetic analysis. We used the newly cultivated bottlenose dolphin herpesvirus (TTHV) to develop a direct enzyme-linked immuno-sorbent assay for anti-TTHV antibodies in banked sera of these dolphins. The percentage of positive samples was higher in adults (20/21, 95%) than in juveniles (7/15, 47%). Seroconversion occurred around the age of onset of sexual behavior. Although herpesvirus infection has been associated with abortion, perinatal mortality, and urogenital neoplasia in other species, we found no evidence of herpesvirus infection by PCR in tissues from six cases of abortion and perinatal mortality, and no diagnoses of urogenital tumors in 24 bottlenose dolphins from this zoo collection that died since 1990. Together, we here report the first successful cultivation from bottlenose dolphins of a herpesvirus that probably causes benign genital plaques, is endemic in this group of dolphins, and is likely transmitted by sexual contact.