Instream barriers contribute to population isolation of a small‐bodied, benthic, headwater‐specialist fish (Percidae).

Genetic differentiation in aquatic systems is often driven by geographic distance (isolation by distance) due to the linear and hierarchical distribution of populations, but habitat fragmentation often exacerbates this effect by decreasing population connectivity, leading to isolation by resistance. Stronghold populations of the Kentucky Arrow Darter (Etheostoma spilotum) in the South Fork Kentucky River system within the Daniel Boone National Forest of eastern Kentucky, USA have a high population structure not explained by distance alone. Higher than expected levels of genetic differentiation among proximate populations were hypothesized to be driven by land‐use change, but this was not previously tested. Here we use a riverscape genetics approach to test for the effects of natural landscape features including slope, elevation and stream size, and anthropogenically altered habitat features, including specific conductance (conductivity), culverts and forest cover, on population connectivity and genetic diversity of E. spilotum. We found isolation of populations among all tributary systems and a strong, positive relationship between genetic and geographic distances as expected. However, high conductivity levels due to surface coal mining best explain the population structure observed. We also found signatures of low genetic diversity overall and indicators that culverts may limit upstream movements of E. spilotum. This study provides a novel fine‐scale view of the effects of instream and landscape features on connectivity among, and genetic diversity within populations of an imperilled, small‐bodied, benthic fish. [ABSTRACT FROM AUTHOR]