Urban developments and highways can isolate wildlife populations, subsequently decreasing their numbers and increasing matings between close relatives, which reduces genetic diversity. Low diversity can lead to genetic disorders and lower fitness, known as inbreeding depression. The mountain lion population in California’s Santa Monica Mountains (SMM) is isolated, increasing their risk of inbreeding and inbreeding depression. Inbreeding is traditionally measured with pedigree-based methods, which can underestimate inbreeding when founders are themselves partially inbred. To address this limitation, we conducted a genomic study to assess inbreeding directly based on runs of homozygosity (ROH) in >100 individual mountain lions collected 2002–2024 from the SMM and adjacent areas. We used genotyping-by-sequencing to estimate inbreeding from both pedigrees and ROH. Our analysis revealed that ROH consistently provided higher estimates of inbreeding compared to pedigree-based methods. This study has implications for the conservation of this vulnerable population, already showing physical signs of potential inbreeding depression, similar to the Florida Panthers. The results are crucial for mountain lion conservation, particularly in light of the Wallis Annenberg Wildlife Crossing, and data generated establishes a baseline for assessing this population’s future genetic health and fitness. This information is vital for understanding factors contributing to effective conservation planning.