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Biodiversity is being lost at an unprecedented rate, and resources for conservation efforts are limited. This is particularly problematic in the Great Plains of North America, where land-cover conversion for agriculture and energy production has reduced habitat for many species. In the U.S. portion of the Great Plains, a growing human population and a concomitant increasing need for food, fiber, and energy have caused landscape transformations that have resulted in over 700 vertebrate species currently being listed by state and federal conservation agencies as being at-risk in this region. Conservation efforts for such a large number of species will be most efficient when applied to areas with large numbers of these species, but such areas have never before been identified. We overlaid range maps created by the U.S. Geological Survey's Gap Analysis Program for terrestrial vertebrate species to identify hotspots of high concentrations of U.S. state-defined Species of Greatest Conservation Need (SGCN; species that have identified as being rare or otherwise vulnerable enough to warrant conservation action in a given state) in the short-and mixed-grass prairie ecoregions of the southern and central Great Plains of the United States. We identified hotspots for species currently listed as SGCN as well as those pending designation, and a combined (current and pending) group. We then used data from the U.S. Geological Survey's Protected Areas Database on land ownership and from the U.S. Department of Agriculture's National Agricultural Statistics Service on land use/land cover to quantify the types of land ownership and land use/land cover types in hotspots to give land managers necessary information to address conservation of at-risk species in the Great Plains. Sufficient data were present for examination of 289 at-risk terrestrial vertebrate species. Hotspots of these species were located mostly on state-or federally-managed land in eastern New Mexico, Colorado, and west Texas. The current hottest hotspots were associated with areas with more natural/less anthropogenic forms of land use/land cover; areas with the lowest numbers of SGCNs had proportionately more cropland and less grassland than did hotspots. Identifying regional hotspots of at-risk biodiversity, and describing land use/land cover features associated with such areas, offers an opportunity to take a multi-species approach in more precisely establishing areas of conservation concern in the U.S.