Upper respiratory tract disease (URTD) in the gopher tortoise (Gopherus polyphemus) is highly contagious and has been implicated in the reduction of populations throughout the range. With the exception of a few limited studies, the prevalence of URTD in Georgia, USA tortoise populations is poorly known. We found that exposure to Mycoplasma agassizii and Mycoplasma testudineum, associated with URTD, varied geographically among 11 Georgia tortoise populations. The prevalence of antibodies to M. agassizii in individual populations was either very low (0–3%, n = 7 populations) or very high (96–100%, n = 4 populations), whereas there was variation in the prevalence of antibodies to M. testudineum among populations (20–61%, n = 10) with only one site being negative. Five sites had tortoises with antibodies to both pathogens, and these were the only sites where we observed tortoises with clinical signs consistent with URTD. We did not find tortoises with clinical signs of URTD at sites with tortoises with antibodies only to M. testudineum, which provides evidence that this organism may be of limited pathogenicity for gopher tortoises. Collectively, these data indicate that both M. agassizii and M. testudineum are present in Georgia populations of gopher tortoises and that clinical disease is apparent in populations where both pathogens are present. Additional research is needed to better understand the role of these two pathogens, and other potential pathogens, in the overall health of tortoise populations, especially if future conservation efforts involve translocation of tortoises.