The slender chub (Erimystax cahni) is a federally threatened fish native to and geographically restricted to eastern North America. More specifically, the Powell, Clinch, and lower Holston Rivers in Tennessee were historical collection areas. Habitat degradation from multiple sources, including surface mining, agriculture, dams, and urbanization, is associated with the decline of this species - an obligate inhabitant of gravel shoals in large rivers. As of 1964, only 15 voucher specimens were known and no living specimens were documented for decades. A federal recovery plan and Species Status Assessment were developed to determine if slender chub still exists and could be restored. Given the lack of recent observations using conventional sampling to search for its presence, we used environmental DNA sampling to determine their potential presence. Our specific objectives were to (1) develop a quantitative polymerase chain reaction (qPCR) assay aimed at species-specific detection and (2) sample historically known areas of collection. We sampled 43 sites in the Clinch and Powell Rivers. For the first time in almost two decades, we provide evidence for the continued existence of a putatively extinct species. We detected evidence of E. cahni in both the Clinch and Powell Rivers, but only at a few sites. We cannot confirm the presence of E. cahni, and positive eDNA matches could be attributed to amplification from a hybrid. Our results do indicate slender chub DNA perseveres in the Clinch and Powell Rivers and can inform resource agencies of localities to pursue on-the-ground searches for the slender chub with conventional methods (e.g., snorkeling) and potential restoration and recovery sites.