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ABSTRACT Restoring connectivity via assisted migration is a useful but currently underused approach for maintaining genetic diversity and preventing extirpations of threatened species. The use of assisted migration as a conservation strategy may be limited by the difficulty of balancing the benefits of reconnecting populations (including reduced inbreeding depression and increased adaptive capacity) with the perceived risk of outbreeding depression, which requires comprehensive knowledge of the landscape of adaptive, neutral, deleterious, and structural variation across a species' range. Using a combination of reduced‐representation and whole‐genome sequencing, we characterized genomic diversity and differentiation for the Arkansas Darter (Etheostoma cragini) across its range in the Midwestern US. We found strong population structure and large differences in genetic diversity and effective population sizes across drainages. The strength of genetic isolation by river distance differed among drainages, with landscape type surrounding streams and impoundments also contributing to genetic isolation. Despite low effective population sizes in some populations, there was surprisingly little evidence for recent inbreeding (based on the absence of long runs of homozygosity) or for elevated levels of deleterious variation in smaller populations. Considering neutral, adaptive, deleterious, and structural variation allowed us to identify several potential recipient populations that may benefit from translocations and potential donor sites throughout the range. Planning translocation strategies intended for restored connectivity and possible genetic rescue at earlier stages in species decline will likely increase the probability of retaining genetic diversity and population persistence over the long term while minimizing risks associated with translocation.