Fish assemblages in rivers of the Midwestern United States are an important component of the region's natural resources and biodiversity. We characterized the physical environment and presence of dams in a series of reaches in three eastern Iowa rivers tributary to the Mississippi River and related these characteristics to the fish assemblages present. Some physical characteristics were similar among the 12 study reaches, whereas others differed substantially. We found a total of 68 species across the 12 study reaches; 56 in the Turkey River, 51 in the Maquoketa River and 50 in the Wapsipinicon River. Seventeen species could be described as ‘downstream-distributed’; 15 being found only in the lowest reach of one or more rivers and the other two being found only in the lowest reaches or two or more contiguous reaches including the lowest reach. Two species could be described as ‘upstream-distributed’, being found only in an uppermost reach. Non-metric multidimensional scaling ordination illustrated similarities among reaches, and five physical variables were significantly correlated with assemblage similarities. Catchment area and number of dams between reaches and the Mississippi River were strongly correlated with assemblage similarities, but the directions of their effects were opposite. Catchment area and number of dams were confounded. The collective evidence to date suggests that the pervasiveness of dams on rivers significantly alters fish assemblages, making underlying patterns of species change and relationships with naturally varying and human-influenced physical characteristics along a river's course difficult to discern.