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Fish and benthic macroinvertebrate assemblages often provide insight on ecological conditions for guiding management actions. Unfortunately, land use and management legacies can constrain the structure of biotic communities such that they fail to reflect habitat quality. The purpose of this study was to describe patterns in fish and benthic macroinvertebrate assemblage structure, and evaluate relationships between biota and habitat characteristics in the Chariton River system of south-central Iowa, a system likely influenced by various potential management legacies (e.g., dams, chemical removal of fishes). We sampled fishes, benthic macroinvertebrates, and physical habitat from a total of 38 stream reaches in the Chariton River watershed during 2002–2005. Fish and benthic macroinvertebrate assemblages were dominated by generalist species tolerant of poor habitat quality; assemblages failed to show any apparent patterns with regard to stream size or longitudinal location within the watershed. Metrics used to summarize fish assemblages and populations [e.g., presence–absence, relative abundance, Index of Biotic Integrity for fish (IBIF)] were not related to habitat characteristics, except that catch rates of piscivores were positively related to the depth and the amount of large wood. In contrast, family richness of benthic macroinvertebrates, richness of Ephemeroptera, Trichoptera, and Plecoptera taxa, and IBI values for benthic macroinvertebrates (IBIBM) were positively correlated with the amount of overhanging vegetation and inversely related to the percentage of fine substrate. A long history of habitat alteration by row-crop agriculture and management legacies associated with reservoir construction has likely resulted in a fish assemblage dominated by tolerant species. Intolerant and sensitive fish species have not recolonized streams due to downstream movement barriers (i.e., dams). In contrast, aquatic insect assemblages reflected aquatic habitat, particularly the amount of overhanging vegetation and fine sediment. This research illustrates the importance of using multiple taxa for biological assessments and the need to consider management legacies when investigating responses to management and conservation actions.