In the mid-1990s, the population size of Florida panthers became so small that many individuals manifested traits associated with inbreeding depression (e.g., heart defects, cryptorchidism, high pathogen-parasite load). To mitigate these effects, pumas from Texas were introduced into South Florida to augment genetic variation in Florida panthers. In this study, we report a de novo puma genome assembly and annotation after resequencing 10 individual genomes from partial Florida-Texas-F₁ trios. The final genome assembly consisted of ∼2.6 Gb and 20,561 functionally annotated protein-coding genes. Foremost, expanded gene families were associated with neuronal and embryological development, whereas contracted gene families were associated with olfactory receptors. Despite the latter, we characterized 17 positively selected genes related to the refinement of multiple sensory perceptions, most notably to visual capabilities. Furthermore, genes under positive selection were enriched for the targeting of proteins to the endoplasmic reticulum, degradation of mRNAs, and transcription of viral genomes. Nearly half (48.5%) of ∼6.2 million SNPs analyzed in the total sample set contained putative unique Texas alleles. Most of these alleles were likely inherited to subsequent F₁ Florida panthers, as these individuals manifested a threefold increase in observed heterozygosity with respect to their immediate, canonical Florida panther predecessors. Demographic simulations were consistent with a recent colonization event in North America by a small number of founders from South America during the last glacial period. In conclusion, we provide an extensive set of genomic resources for pumas and elucidate the genomic effects of genetic rescue on this iconic conservation success story.