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The alligator snapping turtle (Macrochelys temminckii) is the largest freshwater turtle species in North America and is classified as state endangered in Illinois. Head start programs that include health assessments and pathogen detection are being conducted to restore this species to its historic range. Physical examinations and oral and cloacal swabs were collected from 97 head start alligator snapping turtles prerelease and from 58 recaptured turtles postrelease in 2014. Conventional polymerase chain reaction and sequencing targeting the RNA polymerase beta subunit gene and the 16S-23S ribosomal RNA intergenic spacer detected a single prerelease individual with Mycoplasma sp. with closest relation to Mycoplasma spp. of other freshwater turtles. This individual did not display any clinical signs at the time of release or sampling. Mycoplasmosis has been characterized as a disease of conservation concern in tortoises and may represent a threat to small and fragmented chelonian populations. Findings of this study indicate that Mycoplasma sp. DNA is present in an Illinois alligator snapping turtle, and future investigation into the impact of this infection may support conservation efforts. [ABSTRACT FROM AUTHOR]