We collected fishes at 28 sites of the middle Wabash River, Indiana, using a boat electrofisher and at ten additional sites using a 10-m beach seine. We used Canonical Correspondence Analysis to test for fish assemblage variation that was explained by variation in water depth and substrate frequency. The multivariate analyses resulted in a longitudinal gradient in water depth and frequency of occurrence of substrate size categories that explained a large component of variation among fish assemblages for both sampling regimes. Our results illustrate that substrate composition and water depth variation are significant environmental predictors of fish assemblage composition for a large river such as the Wabash River.