On a global scale biodiversity within river networks is threatened by interactions between habitat fragmentation and altered hydrologic regimes. In the Great Plains of North America, stream networks are fragmented by >19,000 anthropogenic barriers and flow regimes are altered by surface water retention and groundwater extraction. We documented the distribution of anthropogenic barriers and dry stream segments in five basins covering the central Great Plains to assess effects of broad-scale environmental change on stream fish community structure, distribution of reproductive guilds, and genetic integrity of select populations. We used an information-theoretic approach to rank competing models involving fragmentation, discharge magnitude, and percent of time streams had zero flow (i.e., desiccation) to test effects of environmental alterations on fish communities, guilds, and genes. Fragmentation caused by anthropogenic barriers was most common in the eastern Great Plains, but stream desiccation became more common to the west where rivers are underlain by the depleted (i.e., extraction > recharge) High Plains Aquifer. Longitudinal gradients in fragmentation and desiccation contributed to spatial shifts in community structure from diverse communities dominated by pelagic reproductive guilds where fragmentation and desiccation were least, to homogenized communities dominated by benthic guilds where fragmentation and desiccation were common. Modeling results revealed these shifts were primarily associated with decline of pelagic reproductive guilds, notably small-bodied pelagophilic and lithopelagophilic fishes that declined in association with decreased fragment length and increased number of days with zero flow. Genetic analyses revealed little response in genetic diversity indices among fragments with varying lengths, discharge magnitudes, or percent of time with zero flows for Plains Minnow (Hybognathus placitus), Emerald Shiner (Notropis atherinoides), and Red Shiner (Cyprinella lutrensis). These results suggest that demographic processes far out-weight the effects of genetic constraints (e.g., inbreeding depression, accumulation of deleterious genes) in determining population persistence. Based on our data and considering Plains Minnow to be representative of small-bodied pelagophilic fishes, persisting populations might be used as sources for repatriation efforts assuming said repatriations occur within the boundaries of major river drainages. Patchbased graphs illustrated particular stream fragments characterized by greater lengths, discharge magnitudes sufficient to avoid desiccation, and persistence of small-bodied pelagophilic and lithopelagophilic fishes that might be prioritized for maintenance to enhance conservation of declining guilds. Furthermore, graph theory combined with a barrier prioritization approach revealed specific fragments that could be reconnected to allow recolonization of currently unoccupied fragments with the mitigation or removal of small dams (10 m height). Our study represents one of the most comprehensive assessments of fish diversity responses to broad-scale environmental change in the Great Plains and is useful for natural resource managers charged with halting (e.g., maintaining current diversity) or reversing (e.g., recolonizations or reintroductions) the prevailing pattern of declining fish diversity in the Great Plains.