Etheostoma kantuckeense and E. lawrencei are former members of the wide ranging E. spectabile species complex. Etheostoma kantuckeense is endemic to the Barren River Basin in Southern Kentucky and Northern Tennessee, while E. lawrencei occurs in the Green River, Salt River, and Cumberland River Basins of Central and Eastern Kentucky. Isolation of populations within these drainages has allowed for a relatively recent evolutionary divergence, leading to slight differences in morphology. This study was conducted to address if geographical isolation has led to measurable differences in the ecology of these two species. In particular, habitat preference across three spatial scales and growth rates were examined. To assess stream preference within a drainage, 59 streams were sampled for fish abundance and environmental parameters in the Upper Barren River Basin (E. kantuckeense; N=24) and the Upper Green River Basin (E. lawrencei; N=35). Channel unit preference and growth rates were compared in two physically similar upland streams, which were sampled monthly from August 2007 through August 2008. Surveys for microhabitat preference were conducted in these same streams in November 2008. Results from a canonical correspondence analysis show that both species occupy equivalent stream types in their respective watersheds. Within the two survey streams, run habitat was preferred over riffle and pool habitats over the course of the year, and both species exhibited similar seasonal habitat shifts. Microhabitat associations for both species were predominately for small to coarse gravel (1–40mm diameter) substrates. In addition, growth of individuals in their first year of life was similar for both species. These results suggest that these species maintain similar ecological traits in their respective watersheds. The retention of these headwater adapted traits in disjunct populations likely promoted vicariant allopatric speciation in these fishes through isolation and the inability to disperse across ecological barriers.