Rate of global biodiversity loss increased significantly during the 20th century associated with human environmental alterations. Specifically, mismanagement of freshwater resources contributed to historical and contemporary loss of stream-dwelling fish diversity and will likely play a role in determining the persistence of species in the future. We present a mechanistic pathway by which human alteration of streams has caused the decline of a unique reproductive guild of Great Plains stream-dwelling fishes, and suggest how future climate change might exacerbate these declines. Stream fragmentation related to impoundments, diversion dams and stream dewatering are consequences of increasing demand for freshwater resources and have effectively created a mosaic of large river fragments throughout the Great Plains of central North America. We analyzed community composition, species population status, fragment size and flow regime components for 60 stream fragments spanning the latitudinal range of the contiguous United States. Stream fragment lengths were a strong predictor of conservation status among pelagic-spawning cyprinid populations, explaining 71% of cumulative extirpations. Mean fragment lengths were least for extirpated (140 ± 55 km) and declining (205 ± 65 km) populations and highest for stable (425 ± 185 km) populations. Similarly, components of flow regimes within fragments associated with magnitude of discharge explained 29% of variation among daily streamflow values, and extirpations were positively correlated (r = 0.36, P = 0.02) with declining discharges. Future climate change scenarios project stream fragments in the southern Great Plains may lose up to 12% of their discharge before 2060, while stream fragments in the northern Great Plains may gain up to 5%. Continued human demand for water resources combined with reduced availability in the southern Great Plains will likely contribute to increased need for fragmentation (e.g., impoundments) and cause further disparity from natural flow regimes. Conservation measures that restore connectivity of river fragments and natural flow regimes will likely benefit pelagic-spawning cyprinids. Moreover, maintenance of long stream fragments will ensure that stable populations of these species do not undergo further declines.