In this paper, we evaluate relationships between in-stream habitat, water chemistry, spatial distribution within a predominantly agricultural Midwestern watershed and geomorphic features and fish assemblage attributes and abundances. Our specific objectives were to: (1) identify and quantify key environmental variables at reach and system wide (watershed) scales; and (2) evaluate the relative influence of those environmental factors in structuring and explaining fish assemblage attributes at reach scales to help prioritize stream monitoring efforts and better incorporate all factors that influence aquatic biology in watershed management programs. The original combined data set consisted of 31 variables measured at 32 sites, which was reduced to 9 variables through correlation and linear regression analysis: stream order, percent wooded riparian zone, drainage area, in-stream cover quality, substrate quality, gradient, cross-sectional area, width of the flood prone area, and average substrate size. Canonical correspondence analysis (CCA) and variance partitioning were used to relate environmental variables to fish species abundance and assemblage attributes. Fish assemblages and abundances were explained best by stream size, gradient, substrate size and quality, and percent wooded riparian zone. Further data are needed to investigate why water chemistry variables had insignificant relationships with IBI scores. Results suggest that more quantifiable variables and consideration of spatial location of a stream reach within a watershed system should be standard data incorporated into stream monitoring programs to identify impairments that, while biologically limiting, are not fully captured or elucidated using current bioassessment methods.