Upper respiratory tract disease (URTD), caused by Mycoplasma agassizii, has been deemed a threat to populations of Mojave Desert Tortoises, Gopherus agassizii. Previous work on URTD has focused on serology and visual health examinations to determine the extent of this disease in some natural tortoise populations. Here, we present the first range-wide study of the presence of the pathogen, M. agassizii, in Mojave Desert Tortoises. We detected M. agassizii in tortoise populations throughout the Mojave Desert, with notable differences in prevalence of M. agassizii among sampling sites within tortoise genotypes and sampling years. Analyses of three genetic markers in the M. agassizii genome indicated very low nucleotide diversity and no relevant spatial structuring of Mycoplasma haplotypes. We use published lines of evidence to discuss the roles of rare transmission events and long-term mycoplasmal persistence in individual hosts on tortoise URTD dynamics.