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Cryptosporidium spp. oocysts are transmitted via a fecal-oral route, e.g., water sources, and the parasite is a leading cause of waterborne disease in the United States. Research into Cryptosporidiosis in rural areas is lacking. This study utilized eDNA methods to determine if Cryptosporidium spp. was present in Harlan, Kentucky, an underserved area in Appalachia. We used a pump-powered commercially available Smith-Root Citizen Scientist eDNA (Vancouver, WA) system to filter freshwater sources and collect eDNA. Samples were collected from tap and spring water as controls and three times from five locations on the three tributaries of the Cumberland River: Martins Fork River, Poor Fork River, and Clover Fork River. DNA was extracted from the filter paper and a genus specific Cryptosporidium PCR was utilized to determine the prevalence of Cryptosporidium. Seventy-seven percent (n=43) of samples were PCR-positive and negative controls did not amplify. Sanger sequencing is ongoing to confirm the PCR findings and determine Cryptosporidium species. These data may be informative for public health in Appalachia by identifying potential risks along accessible waterways.