We evaluated cause of injury and quantified levels of three potential mycoplasmal pathogens (Mycoplasma agassizii, Mycoplasma testudineum, and an emydid mycoplasma) in three-toed box turtles (Terrapene carolina triunguis) from the greater St. Louis, Missouri, US area, brought to and housed at the Wildlife Rescue Center (Ballwin, Missouri, US) in 2015 and 2016. We created a probebased quantitative PCR (qPCR) assay for the emydid mycoplasma, with a similar specificity and sensitivity as the existing qPCR assays for M. agassizii and M. testudineum. All three microbes have been implicated in the development of upper respiratory tract disease in turtles and tortoises. We assessed whether signs of respiratory disease, sex, type of trauma, or treatment (administration of antibiotics) affected the presence of pathogens. We found that the most common types of injury experienced by turtles (n=85) were due to motor vehicles and other types of machinery, and that injuries due to motor vehicles were the most severe. We found a 61% prevalence of emydid mycoplasma (n=28) but M. agassizii or M. testudineum were not detected. Prevalence of disease and antibiotic treatment was too low to statistically relate to levels of mycoplasma. Sex and type of trauma were not associated with levels of emydid mycoplasma. The box turtle population we sampled did not experience signs of respiratory disease due to the fairly widespread prevalence of emydid mycoplasma. However, mycoplasmal diseases can be pathogen load-dependent. The qPCR we designed can be used to assess levels of emydid mycoplasma in other emydid species, populations, and individuals, in which there might be a positive association between the microbe and expression of respiratory disease.