Urban development and roads, especially freeways, can isolate mammal populations, increasing the risk of low genetic diversity through genetic drift and inbreeding (i.e., matings between closely related individuals). This low diversity can result in genetic disorders and a decline in fitness known as inbreeding depression. The mountain lion population in California's Santa Monica Mountains (SMM) has become increasingly isolated due to human activities. To evaluate the effects of isolation, we conducted genotyping-by-sequencing of >100 individual mountain lions collected by the National Park Service from the SMM and adjacent Simi Hills and Santa Susana Mountains (2002–2023), and employed both pedigree-based and direct estimation methods to infer levels of inbreeding. This research has significant conservation implications for this vulnerable population, particularly as ongoing urban expansion threatens habitat connectivity. This study is particularly relevant and timely as the Wallis Annenberg Wildlife Crossing is due for completion in 2025. This wildlife crossing was designed to increase connectivity for this and other populations, and the data generated from this study establishes a critical baseline for assessing this population's future genetic health and fitness, offering insights that are essential for effective conservation planning. |