Although benthic insectivorous fishes such as darters and sculpins represent a significant component of riffle communities, few studies have compared the habitat use of these non-related but ecologically similar fishes. The objectives of this study were to examine the habitat use of Etheostoma olmstedi (tessellated darter) compared to Cottus bairdi (mottled sculpin) in Nescopeck Creek and Cottus cognatus (slimy sculpin) in Jack's Creek, Pennsylvania through underwater observation. Etheostoma olmstedi occupied habitats with significantly deeper waters than those available, whereas adult and young of the year Cottus occupied habitats with significantly faster water velocities than those available. Canonical discriminant analysis revealed microhabitat partitioning between E. olmstedi and each Cottus species. Cottus bairdi and C. cognatus occupied significantly shallower habitats with faster water velocities than E. olmstedi. Sculpin species were observed most frequently under substrate whereas E. olmstedi occurred most frequently on the top surface of the substrate. Hurlbert's standardized niche breadth values indicated that C. bairdi and C. cognatus were habitat specialists with regard to water velocity measures, but exhibited generalistic patterns of depth and substrate size use. Etheostoma olmstedi was a habitat specialist with respect to depth, but exhibited generalistic patterns of resource use for substrate size. Differential habitat use by these benthic fishes is consistent with the hypothesis that resource partitioning facilitates species coexistence among stream fishes.