Erimystax cahni (Slender Chub) is a federally threatened fish native to the Powell, Clinch, and lower Holston rivers of Tennessee and Virginia. No individuals have been captured or reported for over 2 decades. We developed a species-specific environmental DNA (eDNA) assay to survey for its presence throughout its native distribution in new and historical sites. We detected Slender Chub eDNA in both the Powell and Clinch rivers in 3 of 43 sites sampled during summer 2022. The presence of live individuals could not be confirmed, and the detection of positive eDNA matches could result from pure individuals or hybrids. Note that positive eDNA matches do not preclude the presence of hybrid individuals; however, they can help guide managers to intensively sample areas with conventional methods (e.g., snorkeling and seining), which could improve the probability of capture of living individuals.