Mycoplasmosis is a transmissible upper respiratory tract disease that has affected plans for management and conservation of wild desert and gopher tortoises in the United States. Although impact of mycoplasmosis on populations of desert and gopher tortoises is unknown, increased prevalence of seropositive animals as well as field observations of clinically ill tortoises have occurred in association with declining populations. In order to help in the identification of potentially infected animals, three tests have been developed to diagnose mycoplasmal infections of tortoises: 1) direct mycoplasmal culture; 2) detection of mycolplasmal chromosomal DNA by polymerase chain reaction (PCR); and 3) detection of anti-Mycoplasma antibodies in tortoise plasma by enzyme-linked immunosorbent assay (ELISA). Each test provides different, complementary information that collectively can be used to define tortoise mycoplasmal infection status. The types of samples required, the predictive value, interpretation, and cost vary among tests. These assays have been used for epidemiological surveys and in decision making for relocation, repatriation, or captive management of tortoises to minimize the risk of outbreaks of mycoplasmal respiratory disease and spread of the causative agent of this disease. Certain features of mycoplasmal infections of tortoises and other animals create a diagnostic dilemma. Multiple Mycoplasma species can cause respiratory disease with identical clinical presentations. Further, individual strains of a given species may vary with respect to their virulence potential, and some species may be commensals rather than pathogens. Current diagnostic tests may not differentiate among mycoplasmal species or strains or permit determination of pathogenicity of individual isolates. Thus, the information provided by testing is not a simple 'positive' vs. 'negative' issue. While these tests provide much needed information on the exposure of tortoise populations to Mycoplasma species, they do not provide a complete picture of the overall health status of individual tortoises or populations. Unfortunately, test results are often used to make life and death decisions concerning disposition of tortoises being displaced by land development without a complete understanding of the limitations of the diagnostic tests or any consideration of other infectious agents that might be present.