Upper respiratory tract disease (URTD) has been associated with major losses of free-ranging desert tortoises (Gopherus agassizii) in the southwestern United States. This prompted a clinical examination of 68 free-ranging desert tortoises for signs of URTD and sampling for Mycoplasma agassizii, the causative agent of URTD. Tortoises were sampled from three sites in the eastern Mojave Desert (1992–93) and from three sites in the Sonoran Desert (1992–94). Plasma was analyzed for antibodies to M. agassizii using an enzyme-linked immunosorbent assay (ELISA). Nasal aspirate from 12 Sonoran tortoises was tested using polymerase chain reaction (PCR) test to detect the 16S rRNA gene of M. agassizii. Nasal aspirate from all tortoises was cultured for Mycoplasma sp. In the Mojave Desert, nine tortoises had clinical signs of URTD (n=28). Eight of the nine tortoises with clinical signs were seropositive for M. agassizii. In the Sonoran Desert, there were no clinical signs of URTD, but two tortoises were seropositive (n=40), and two tortoises had PCR results indicating presence of M. agassizii (n=12). Monitoring of URTD is recommended for Mojave tortoises, and further monitoring is needed for Sonoran tortoises because we do not know the extent of this disease in this population.