Biotic indices (bioindicators) can be individual species, species groups, or communities of species used to assess habitat quality. But, to be used effectively, managers require basic information on species used as indicators, including species distribution, differentiation between similar species, and environmental conditions associated with species presence. We addressed these problems concurrently in two related species, the Mottled Sculpin (Cottus bairdii) and the Slimy Sculpin (Cottus cognatus), as habitat quality indicators in the Manistee River in Michigan, USA. We determined the abundance and distribution of these species and related their presence to concurrent in-stream measurements of temperature, dissolved oxygen, pH, conductivity, turbidity, and stream quality score based on macroinvertebrate diversity. Cladistic analyses of CO1 supported recognition of Mottled Sculpin and Slimy Sculpin as distinct species and confirmed initial field identification to species using morphological characteristics. Both species were most abundant in headwater regions, decreased downstream, and were sympatric at 5 of 12 (42%) locations. Mottled Sculpin were associated with lower conductivity, pH, and stream quality scores. Slimy Sculpin were associated with higher levels of DO and lower levels of turbidity. As a management indicator species of the US Forest Service, Mottled Sculpin alone may be ineffective as a habitat quality indicator, but concurrent use of Mottled Sculpin and Slimy Sculpin as a related-species complex might allow sufficient coverage to permit assessment of stream quality if species-specific differences in environmental tolerances are precisely determined.