Regional assessment of cumulative impacts of dams on riverine fish assemblages provides resource managers essential information for dam operation, potential dam removal, river health assessment and overall ecosystem management. Such an assessment is challenging because characteristics of fish assemblages are not only affected by dams, but also influenced by natural variation and human-induced modification (in addition to dams) in thermal and flow regimes, physicochemical habitats and biological assemblages. This study evaluated the impacts of dams on river fish assemblages in the non-impoundment sections of rivers in the states of Michigan and Wisconsin using multiple fish assemblage indicators and multiple approaches to distinguish the influences of dams from those of other natural and human-induced factors. We found that environmental factors that influence fish assemblages in addition to dams should be incorporated when evaluating regional effects of dams on fish assemblages. Without considering such co-influential factors, the evaluation is inadequate and potentially misleading. The role of dams alone in determining fish assemblages at a regional spatial scale is relatively small (explained less than 20% of variance) compared with the other environmental factors, such as river size, flow and thermal regimes and land uses jointly. However, our results do demonstrate that downstream and upstream dams can substantially modify fish assemblages in the non-impoundment sections of rivers. After excluding river size and land-use influences, our results clearly demonstrate that dams have significant impacts on fish biotic-integrity and habitat-and-social-preference indicators. The influences of the upstream dams, downstream dams, distance to dams, and dam density differ among the fish indicators, which have different implications for maintaining river biotic integrity, protecting biodiversity and managing fisheries.