Habitat loss and fragmentation drive biodiversity decline by reducing connectivity, leading to genetic isolation, loss of diversity, and reduced fitness within populations. Gene flow via translocations can mitigate these issues by introducing individuals to increase diversity, alleviate inbreeding, and improve adaptive capacity—a process known as genetic rescue. However, limited understanding of genetic structure and historic connectivity of populations can hinder its application. We used Stephens’ kangaroo rat (Dipodomys stephensi), a species threatened by habitat loss and fragmentation, as a model for developing range-wide management strategies based on genetic structure and connectivity. We analyzed microsatellite data to investigate historical and contemporary genetic differentiation. Using landscape resistance models, we found that while geographic features explain much of the genetic structure, anthropogenic barriers contribute to current genetic differentiation and are expected to continue driving evolution as populations become increasingly isolated. Population models show that multiple translocations are far more effective than a single translocation at increasing persistence and heterozygosity; varying the frequency and size of translocations was less important than maintaining a management strategy. For small populations, habitat restoration to increase carrying capacity was also required to prevent extirpation. Our findings highlight the value of integrating genetic data into conservation planning.