Linear barriers pose significant challenges for wildlife gene flow, impacting species persistence, adaptation, and evolution. While numerous studies have examined the effects of linear barriers (e.g., fences, roadways) on partitioning urban and non-urban areas, understanding their influence on gene flow within cities remains limited. Here, we investigated the impact of linear barriers on coyote (Canis latrans) population structure in Seattle, Washington, where major barriers (i.e., interstate highways and bodies of water) divide the city into distinct quadrants. Notably, private allele analysis underscored limited interbreeding among quadrants. When comparing one quadrant to the next, there were up to 16 private alleles within a single quadrant, representing nearly 22% of the population allelic diversity. Our analysis revealed weak isolation by distance, and despite being a highly mobile species, genetic structuring was apparent between quadrants even with extremely short geographic distance between individual coyotes, implying that Interstate 5 and the Ship Canal act as major barriers. Lack of gene flow may stem from the perceived risk of crossing these barriers, as even despite the presence of structural connectivity features (e.g., bridges and underpasses), functional connectivity may remain limited. Urban areas provide refuge and resources for wildlife but come with tradeoffs, as evidenced by restricted gene flow and potential long-term impacts on population viability and evolution. This study advances our understanding of gene flow and its consequences in cities, a crucial component for bolstering wildlife conservation and management in rapidly urbanizing environments.