Biodiversity in stream networks is threatened globally 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 and distribution of reproductive guilds. We used an information theoretic approach to rank competing models in which fragmentation, discharge magnitude, and percentage of time streams had zero flow (a measure of desiccation) were included to predict effects of environmental alterations on the distribution of fishes belonging to different reproductive guilds. 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 taxonomically and functionally 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. Graph theory combined with a barrier prioritization approach revealed specific fragments that could be reconnected to allow fishes within these guilds to colonize currently unoccupied fragments with the mitigation or removal of small dams (,10 m height). These findings are useful for natural resource managers charged with halting or reversing the prevailing pattern of declining fish diversity in the Great Plains. Our study represents one of the most comprehensive assessments of fish diversity responses to broad-scale environmental change in the Great Plains and provides a conservation strategy for addressing the simultaneous contributions of fragmentation and flow alteration to the global freshwater biodiversity crisis.