Historical change in fish assemblage structure was evaluated in the mainstems of the Des Moines, Iowa, Cedar, Wapsipinicon, and Maquoketa rivers, in Iowa. Fish occurrence data were compared in each river between historical and recent time periods to characterize temporal changes among 126 species distributions and assess spatiotemporal patterns in faunal similarity. A resampling procedure was used to estimate species occurrences in rivers during each assessment period and changes in species occurrence were summarized. Spatiotemporal shifts in species composition were analyzed at the river and river section scale using cluster analysis, pairwise Jaccard's dissimilarities, and analysis of multivariate beta dispersion. The majority of species exhibited either increases or declines in distribution in all rivers with the exception of several “unknown” or inconclusive trends exhibited by species in the Maquoketa River. Cluster analysis identified temporal patterns of similarity among fish assemblages in the Des Moines, Cedar, and Iowa rivers within the historical and recent assessment period indicating a significant change in species composition. Prominent declines of backwater species with phytophilic spawning strategies contributed to assemblage changes occurring across river systems.