Background <p>The jaguar (<i>Panthera onca</i>) is a keystone species within diverse ecosystems ranging from dense rainforests to open grasslands across Central and South America. However, its populations are declining rapidly due to anthropogenic actions, such as deforestation and poaching. Here we investigate the effects of this decline on genetic diversity and genetic health. Utilizing both modern and historical museum samples, we infer population structure and immunome variability in 25 jaguars to identify unique genetic diversity that can inform targeted conservation efforts.</p> Results <p>Our genome-wide analyses identifies three distinct geographic populations: Central America, South American lowlands, and South American highlands. Modern samples that exhibit lower levels of heterozygosity also show higher levels of inbreeding. The South American lowland population shows the lowest levels of inbreeding, while the highland population exhibits the lowest overall immunome-wide variability. However, the innate (Natural Killer Cell Complex, Toll-Like Receptor) and adaptive (Major Histocompatibility Complex Class II) immune genes, which are crucial for adaptive responses and disease resilience, show high diversity in terms of heterozygosity and haplotype diversity in individuals of all three populations.</p> Conclusions <p>South American highland and Central American jaguars face significant threats from habitat loss and fragmentation. The observed genome- and immunome-wide diversity in historical and modern jaguars reflect their recent demographic decline and challenges of local adaptation. We recommend re-evaluating evolutionarily significant units to prioritize conservation strategies, ensuring the preservation of unique genetic and adaptive diversity crucial for the species’ resilience and long-term survival.</p>