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Epizootic events of pneumonia, presumably caused by Mycoplasma ovipneumoniae, in bighorn sheep (Ovis canadensis) have been observed in the western United States and Canada. Until recently, it was thought that populations of Mexican (O. c. mexicana) and Nelson's (O. c. nelsoni) desert bighorn sheep in Texas, USA, had not been exposed to Mycoplasma. Evidence of disease and potential population decline from outbreaks of M. ovipneumoniae are now known from several populations across the Trans‐Pecos Ecoregion with documented instances of pneumonia and bluetongue in desert bighorn sheep from the Van Horn Mountains and Black Gap Wildlife Management Area. These disease events, especially those in 2019–2021, may be a result of increasing populations of aoudad (Ammotragus lervia), an introduced and invasive ungulate, in the region. With large population sizes and similar movement patterns as desert bighorn sheep, aoudad potentially are the reservoirs for bacterial and viral diseases, such as pneumonia and bluetongue, and are possibly contributing to the decline of desert bighorn sheep. Herein, we optimized the multi‐locus sequence typing (MLST) with modifications in the Taq polymerase and annealing temperatures to determine the genetic identity of Mycoplasma strains or species within the nasal passages of desert bighorn sheep and aoudad in the Trans‐Pecos Ecoregion of Texas. Four loci (small ribosomal unit, 16S; 16S‐23S intergenic spacer region, IGS; RNA polymerase B, rpoB; gyrase B, gyrB) were characterized using MLST. Based on results from the modified MLST technique, we identified 9 desert bighorn sheep and 5 aoudad with M. ovipneumoniae, 9 aoudad with bacterial sequences genetically similar to M. conjunctivae, and 10 aoudad with bacterial sequences genetically similar M. hyopneumoniae. Of these, 9 aoudad possessed bacterial sequences genetically similar to both M. conjunctivae and M. hyopneumoniae. Among the 4 diagnostic loci, genetic divergence of M. ovipneumoniae ranged from 0.00–0.90% among desert bighorn sheep and aoudad. Future sampling efforts of seemingly asymptomatic aoudad, and asymptomatic, visibly sick, or deceased desert bighorn sheep, are important to monitor the spread of disease in desert bighorn sheep populations across mountain ranges in western Texas. It is imperative that aoudad removal plans are implemented to reduce and eliminate current infections and putative transmission of M. ovipneumoniae, prevent future disease outbreaks of pneumonia, and ultimately conserve desert bighorn sheep for future generations. [ABSTRACT FROM AUTHOR]