Pumas (Puma concolor; also known as mountain lions and cougars) in southern California live among a burgeoning human population of roughly 20 million people. Yet little is known of the consequences of attendant habitat loss and fragmentation, and human-caused puma mortality to puma population viability and genetic diversity. We examined genetic status of pumas in coastal mountains within the Peninsular Ranges south of Los Angeles, in San Diego, Riverside, and Orange counties. The Santa Ana Mountains are bounded by urbanization to the west, north, and east, and are separated from the eastern Peninsular Ranges to the southeast by a ten lane interstate highway (I-15). We analyzed DNA samples from 97 pumas sampled between 2001 and 2012. Genotypic data for forty-six microsatellite loci revealed that pumas sampled in the Santa Ana Mountains (n = 42) displayed lower genetic diversity than pumas from nearly every other region in California tested (n = 257), including those living in the Peninsular Ranges immediately to the east across I-15 (n = 55). Santa Ana Mountains pumas had high average pairwise relatedness, high individual internal relatedness, a low estimated effective population size, and strong evidence of a bottleneck and isolation from other populations in California. These and ecological findings provide clear evidence that Santa Ana Mountains pumas have been experiencing genetic impacts related to barriers to gene flow, and are a warning signal to wildlife managers and land use planners that mitigation efforts will be needed to stem further genetic and demographic decay in the Santa Ana Mountains puma population.