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Leptospirosis is a water borne zoonotic disease of global significance that is caused by pathogenic species of the genus Leptospira. Pathogenic leptospires live in the kidneys of reservoir or infected animals and are shed in their urine contaminating water, soil, etc. Rodents are considered the primary reservoir of leptospirosis, but little is known about the role of herpetofauna (non-avian reptiles and amphibians) in the epidemiology of the disease. To address this, various species of amphibians and reptiles in the Cumberland Gap Region of the Central Appalachia were screened for the presence of Leptospira spp. Kidneys harvested from of a total of 116 amphibians and reptiles belonging to seven species of snakes, seven species of salamanders, seven species of frogs/toads, seven species of turtles and one species of lizards were tested using a highly specific TaqMan based qPCR that targets lipl32 gene of pathogenic Leptospira spp. Overall, 15 of the tested 116 amphibians and reptiles were positive (12.9%; 95% CI: 7.4%-20.4%). Of the 101 amphibians, 11 were positive (10.9%; 95% CI: 5.6%-18.7%), and 4 of the 15 reptiles tested positive (26.7%; 95% CI: 7.8%-55.1%). The amplified gene fragments of lipl32 from qPCR positive kidneys were sequenced and found to be identical with known pathogenic Leptospira spp. These results suggest that although the proportion of reptiles and amphibians transmitting pathogenic Leptospira spp. within the environment may be low as compared to rodents, they pose a risk to other susceptible hosts that share their habitats and may have role in maintaining a baseline infection in the environment., (© 2022 Wiley-VCH GmbH.)