Roadway infrastructure is a dominating feature of landscapes across the globe and the ecological and evolutionary impacts of these roadways are leading to declines in many natural populations. To date, research on the genetic impacts of roadways on bird populations has produced mixed results, with some studies revealing significant correlations between roads and genetic differentiation and others failing to resolve a significant relationship. Here, we generate a whole-genome dataset to quantify the impacts of roadway infrastructure on the population genetic differentiation of California quail (Callipepla californica), a ground-dwelling and ground-nesting bird species. We non-lethally sampled wild quail from populations located on either side of major and minor roadways in Southern California, a region with the densest road network in the United States. Using a variety of landscape genetic and statistical models, we demonstrate that roadways are the most important factor shaping patterns of genomic differentiation in southern California quail populations. We also show that quail populations sampled alongside the two busiest roadways traversing California have higher levels of inbreeding than other populations within the region, likely resulting from close kin matings within the last 10 generations. Results of this study highlight the importance of roads as major disturbances to genetic connectivity of wild populations and can help inform regional connectivity conservation efforts, including the construction of the Wallis Annenberg Wildlife Crossing over the US 101 freeway.