Within the upper Ohio River watershed, 3 Etheostoma darter species in the subgenus Nothonotus have been documented in disjunct populations and were listed as threatened or endangered in Ohio and Pennsylvania. Tailwater habitat below navigation lock and dam (L/D) installations has been shown to contain diverse darter assemblages. Etheostoma camurum (Bluebreast Darter), E. maculatum (Spotted Darter), and E. tippecanoe (Tippecanoe Darter) often live in similar habitats; thus, we hypothesized that all 3 were occupying tailwater habitat below navigational L/Ds. Electrified benthic trawling verified Bluebreast Darter and Tippecanoe Darter below 8 L/D installations and at water depths varying from 1.4 m to 4.5 m and 1.4 m to 5.9 m, respectively. Spotted Darter was only found below 1 L/D. In the Ohio River, benthic trawling documented Bluebreast Darter and Tippecanoe Darter utilizing habitat located within deposition zones and areas above and below islands. Analysis of contemporary and historic distribution data shows that Bluebreast Darter and Tippecanoe Darter now span large sections of the river, but the range of Spotted Darter is more limited and warrants close monitoring. Our study confirms the effectiveness of utilizing benthic trawling in non-wadeable rivers to survey for benthic species such as river-inhabiting darters.