Etheostoma striatulum (Striated Darter) is an increasingly rare darter found in the middle-to-upper regions of the Duck River watershed in Tennessee. This rarity, coupled with a lack of collection and sampling, has put them in review for listing under the Endangered Species Act. Because of the Striated Darters' reclusive and cryptic behavior, conventional techniques tend to be less effective for detection, requiring more precise and sensitive methods. To provide information regarding distribution and occupancy within the Duck River, we used molecular detection of species-specific environmental DNA to test for presence of the species at historical sampling sites. Assessments indicate that Striated Darter is persisting in all tributaries of historical occurrence, with the addition of 4 new tributaries. However, given that 16 sites produced positive signals for less than a third of qPCR replicates, it appears that 60% of these populations are persisting at low detection levels. Detection was positively correlated with turbidity, which could be explained by their preference for lower-velocity habitats. Our results will assist with identifying critical-habitat locations for the update on the status of Striated Darter while also aiding in the standardization of eDNA-assay development for rare and cryptic species.