Following field observations of wild Agassiz’s desert tortoises (Gopherus agassizii) with oral lesions similar to those seen in captive tortoises with herpesvirus infection, we measured the prevalence of antibodies to Testudinid herpesvirus (TeHV) 3 in wild populations of desert tortoises in California. The survey revealed 30.9% antibody prevalence. In 2009 and 2010, two wild adult male desert tortoises, with gross lesions consistent with trauma and puncture wounds, respectively, were necropsied. Tortoise 1 was from the central Mojave Desert and tortoise 2 was from the northeastern Mojave Desert. We extracted DNA from the tongue of tortoise 1 and from the tongue and nasal mucosa of tortoise 2. Sequencing of polymerase chain reaction products of the herpesviral DNA-dependent DNA polymerase gene and the UL39 gene respectively showed 100% nucleotide identity with TeHV2, which was previously detected in an ill captive desert tortoise in California. Although several cases of herpesvirus infection have been described in captive desert tortoises, our findings represent the first conclusive molecular evidence of TeHV2 infection in wild desert tortoises. The serologic findings support cross-reactivity between TeHV2 and TeHV3. Further studies to determine the ecology, prevalence, and clinical significance of this virus in tortoise populations are needed.