Mycoplasma agassizii (proposed species novum) is the etiologic agent of an upper respiratory tract disease in the desert tortoise (Gopherus agassizii), which is threatened in most of its range. An enzyme-linked immunosorbent assay (ELISA) for the detection of M. agassizii-specific antibodies in desert tortoises was developed with a monoclonal antibody with specificity for desert tortoise immunoglobulin light chain. Plasma samples from one group of tortoises were tested immediately before and 1 month after challenge either with nasal exudate containing M. agassizii or with a purified preparation of M. agassizii. Plasma samples from a second group of known healthy and sick tortoises were also tested. In the first group, the ELISA detected seroconversion in individual tortoises following challenge with M. agassizii. In the second group, ELISA results were positively correlated with the health status of the tortoises, as determined by clinical and pathologic findings. In addition, the ELISA revealed that tortoise antimycoplasma antibodies were specific for M. agassizii when samples were assayed against M. agassizii, M. pulmonis, M. testudinis, and M. gallisepticum antigens. The observed direct correlation between the presence of nasal mucosal lesions and M. agassizii-specific antibodies proved that the ELISA reliably diagnosed M. agassizii infection in desert tortoises and advocates its use for monitoring M. agassizii-induced upper respiratory tract disease in free-ranging desert tortoises.