Across the western Great Plains of North America, groundwater pumping for irrigated agriculture has depleted regional aquifers that sustain stream flow for native fishes. Although declines in Great Plains stream discharge owing to groundwater pumping are widely documented, spatial and temporal patterns in stream intermittency across this broad landscape have yet to be quantified. Successful management of native Great Plains stream fishes into the future will require an explicit understanding of the distribution and abundance of habitats, as well as the connections among those habitats across the region. The goal of this study was to provide critical information and decision-support tools to enhance conservation of threatened fishes in the western Great Plains.