Introduction paragraph/AbstractAcross the geographic range of mountain lions, which includes much of North and South America, populations have become increasingly isolated due to human persecution and habitat loss. To explore the genomic consequences of these processes, we assembled a high-quality mountain lion genome and analyzed a panel of resequenced individuals from across their geographic range. We found strong geographical structure and signatures of severe inbreeding in all North American populations. Tracts of homozygosity were rarely shared among populations, suggesting that assisted gene flow would restore local genetic diversity. However, the genome of an admixed Florida panther that descended from a translocated individual from Central America had surprisingly long tracts of homozygosity, indicating that genomic gains from translocation were quickly lost by local inbreeding. Thus, to sustain diversity, genetic rescue will need to occur at regular intervals, through repeated translocations or restoring landscape connectivity. Mountain lions provide a rare opportunity to examine the potential to restore diversity through genetic rescue, and to observe the long-term effects of translocation. Our methods and results provide a framework for genome-wide analyses that can be applied to the management of small and isolated populations.