The Texas tortoise (Gopherus berlanderi) is one of four native tortoise species found in North America. All four Gopherus species receive some form of government protection because of recent population declines. The Texas tortoise is considered threatened within the state of Texas and is of major conservation interest. As a result of human encroachment into Texas tortoise habitats, there has been an increase in the dissemination of infectious disease into native populations. One of the most important diseases studied in North American tortoises is Mycoplasma agassizii. Seroprevalence studies have been performed in the California desert tortoise (Gopherus agassizii) and Gopher tortoise (Gopherus polyphemus) but have yet to be performed in the Texas tortoise. The purpose of this study was to measure the seroprevalence of Mycoplasma agassizii using an ELISA in two populations of Texas tortoises, a wild population (N = 39) and a population in a wildlife rehabilitation facility (N = 15). Tortoises in the wild population were all seronegative (95% confidence intervals [CI]: 0–7.6%), whereas 80% (12/15; 95% CI: 60–100) of the tortoises from the rehabilitation facility were seropositive. The results of this study suggest that Texas tortoises are exposed to and can mount an immunoglobulin response to Mycoplasma agassizii, which may influence the status of this species in North America. This pilot study suggests that further research is needed to determine the epidemiology of mycoplasmosis in Texas tortoises from the wild and in captivity.