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Mycoplasma species, which can cause upper respiratory tract disease (URTD), are significant pathogens of birds, mammals, fish, and reptiles. Mycoplasmosis is of high concern for chelonian conservation, with the most well-documented cases in gopher and desert tortoises. Mycoplasma sp. infections have been reported in captive and free-living box turtles (Terrapene spp.). We documented URTD associated with Mycoplasma sp. in two free-living, three-toed box turtles (Terrapene carolina triunguis) in Missouri, US. Both turtles were Mycoplasma sp. positive by PCR and had URTD-like clinical signs, including nasal and ocular discharge, palpebral edema, lethargy, and weight loss, during a 6-8-wk period between June and September 2014.