Urban development has major impacts on connectivity among wildlife populations and is thus likely an important factor shaping pathogen transmission in wildlife. However, most investigations of wildlife diseases in urban areas focus on prevalence and infection risk rather than potential effects of urbanization on transmission itself. Feline immunodeficiency virus (FIV) is a directly transmitted retrovirus that infects many felid species and can be used as a model for studying pathogen transmission at landscape scales. We investigated phylogenetic relationships among FIV isolates sampled from five bobcat (Lynx rufus ) populations in coastal southern California that appear isolated due to major highways and dense urban development. Divergence dates among FIV phylogenetic lineages in several cases reflected historical urban growth and construction of major highways. We found strong FIV phylogeographic structure among three host populations north‐west of Los Angeles, largely coincident with host genetic structure. In contrast, relatively little FIV phylogeographic structure existed among two genetically distinct host populations south‐east of Los Angeles. Rates of FIV transfer among host populations did not vary significantly, with the lack of phylogenetic structure south‐east of Los Angeles unlikely to reflect frequent contemporary transmission among populations. Our results indicate that major barriers to host gene flow can also act as barriers to pathogen spread, suggesting potentially reduced susceptibility of fragmented populations to novel directly transmitted pathogens. Infrequent exchange of FIV among host populations suggests that populations would best be managed as distinct units in the event of a severe disease outbreak. Phylogeographic inference of pathogen transmission is useful for estimating the ability of geographic barriers to constrain disease spread and can provide insights into contemporary and historical drivers of host population connectivity.