Human alteration is commonplace among large rivers and often results in changes in the flow regime which can lead to changes in fish community structure. We explored the features of fish community structure, morphological characteristics, functional composition, and life-history attributes in relation to six unique flow regimes in the Missouri and lower Yellowstone rivers where we found significant differences in community composition and abundance. The clearest pattern was the distinction between the channelized portion of the river below the mainstem reservoirs and all other parts of the Missouri and lower Yellowstone rivers due to a marked reduction of species richness above the reservoirs. We also found morphological, functional, and life-history differences among the flow units, with the inter-reservoir communities consisting of slightly more generalist characteristics. Our results suggest some relation between flow and fish community structure, but that human alteration may have the strongest influence in distinguishing community differences in the Missouri and lower Yellowstone rivers.