Adaptive immune diversity is central to population health, yet it is difficult to measure in wildlife using standard genomic datasets. The major histocompatibility complex (MHC) links directly to disease resistance, but its duplicated and variable structure complicates analysis with short-read sequencing, which is the most common and cost-effective approach in conservation genomics. We evaluate whether short-read whole-genome data can reliably recover MHC exons in mountain lions (Puma concolor) in California. Using comparative mapping from the well-annotated domestic cat genome, we identify candidate MHC exon coordinates in puma and then test three recurring pitfalls—reference-mapping bias, collapsed duplicates, and inconsistent callability—across representatives from the state’s ten genetically defined populations. Our objective is to establish a vetted, puma-specific set of “callable” MHC exons and a transparent quality-control workflow suitable for population-scale use. This feasibility study is designed to complement ongoing neutral-genomic work by enabling statewide assessment of adaptive immune diversity and by clarifying what short-read data can—and cannot—capture in complex immune regions. The resulting target set will position future analyses to compare immune diversity among small, isolated populations and larger, more connected ones, informing priorities for long-term monitoring in this wide-ranging carnivore.