Detailed relationships between fish assemblages and habitat and hydrology variation are largely unknown for large rivers. We evaluated hydrology, habitat variation and fish assemblages at 28 sites on the Wabash River, Indiana, USA, during 2005–2008. We calculated hydrologic variation with the Indicators of Hydrologic Alteration software and tested for temporal hydrologic effects on habitat variation of substrate particle size, water depth and water velocity by reducing data into principal component axes that were tested for differences among years with ANOVAs. We then tested for effects of habitat and hydrology variation on fish assemblages with canonical correspondence analysis. These analyses showed significant relationships between hydrologic variation and local habitats, and hydrology and habitat variables had significant relationships with fish assemblage structure. Our Mantel tests resulted in significant concordance among hydrology, local habitat variation and fish assemblage structure, suggesting associations of these variables. These results supported strong connections for hydraulic control over habitat variation and subsequent effects on fish assemblages. Copyright © 2011 John Wiley & Sons, Ltd.