Science-based conservation of riverine fishes can be best targeted with specific information about spatial-ecological controls on the community, including anthropogenic stressors. Because anthropogenic stressors can originate at multiple spatial scales, we investigated the influence of natural and anthropogenic variables summarized within the reach, valley, and catchment on fish community composition along four river mainstems in Ontario, Canada. We used Redundancy Analyses (RDA) to explore models with multi- and single-scale variables on fish community composition. We used partial RDAs to differentiate the relative effects of variable types in multiscale models and to determine if spatial variables explained additional variation in fish community composition. Catchment variables accounted for the majority of explained variation in fish community composition in three of the four rivers, but instream habitat variables accounted for considerable variability in fish community composition in the two rivers that are highly fragmented by dams or naturally occurring rapids. Natural and human-derived fragmentation in rivers may reduce the influence of catchment controls, disrupt longitudinal gradients, and increase the influence of local instream habitat. Environmental variables that explained fish distribution had longitudinal or patchy spatial pattern within rivers, but spatial variables representing impediments to fish dispersal and proximity to receiving waterbodies failed to explain additional variation in fish community composition.