Fragmentation of wildlife populations is increasing on a global scale and understanding current population genetic structure, genetic diversity, and genetic connectivity is key to informing wildlife management and conservation. We genotyped 992 pumas (Puma concolor) at 42 previously developed microsatellite loci and identified 10 genetic populations throughout the states of California and Nevada, USA. Although some genetic populations had large effective population sizes, others were small and inbred. Genetic diversity was extremely variable (heterozygosity, uHe = 0.33–0.53), with some populations nearly as low as an endangered subspecies, the Florida Panther (P. c. coryi, uHe = 0.24). Specifically, pumas in the Sierra Nevada were genetically diverse and formed the largest genetic source population in the region. In contrast, coastal and southern populations surrounded by urbanization had low genetic diversity, fragmented gene flow, and tended to be genetic sinks. The strong population genetic structuring of pumas across California (FST = 0.05–0.39) is vastly different than other genetic studies in less-urbanized states, including our analysis in Nevada, where pumas had few barriers to gene flow and weak population differentiation. Our results have far-reaching conservation and management implications for pumas and indicate large-scale fragmentation in one of North America’s most biodiverse and rapidly-urbanizing regions.