To assess the potential for an epizootic of pneumonia to result from either natural immigration or translocation, we compared the seroprevalence to Mycoplasma ovipneumoniae in several populations of desert bighorn sheep in Arizona. We collected blood samples and nasal or oropharyngeal swabs from 124 desert bighorn sheep (Ovis canadensis nelsoni) from 6 populations in Arizona in 2009 and 2010. M. ovipneumoniae organisms were detected by PCR in 22%, whereas antibodies to M. ovipneumoniae were detected in 47% of tested bighorn sheep. Mycoplasma antibodies were not found in 2 of 6 populations, indicating some bighorn sheep populations in Arizona are naïve to this bacterium. In contrast, others had seroprevalence rates up to 80%. We were able to compare seroprevalence rates and titers over time in 9 individuals (7 individuals included in the 124 bighorn sheep sampled in 2009 and 2010, and 2 individuals originally captured in 2006). Antibody titers persisted for 12 months in individuals from the Kofa National Wildlife Refuge (n = 7) while antibody titers appeared to decline in the Kanab Creek population (n = 2). M. ovipneumoniae is present or has been present in several, but not all, populations of bighorn sheep in Arizona. The results demonstrate the importance of routine health testing for future translocation efforts to reduce disease risk for naive populations.