Spatially heterogeneous landscape factors such as urbanisation can have substantial effects on the severity and spread of wildlife diseases. However, research linking patterns of pathogen transmission to landscape features remains rare. Using a combination of phylogeographic and machine learning approaches, we tested the influence of landscape and host factors on feline immunodeficiency virus (FIV_Lru) genetic variation and spread among bobcats (Lynx rufus) sampled from coastal southern California. We found evidence for increased rates of FIV_Lru lineage spread through areas of higher vegetation density. Furthermore, single-nucleotide polymorphism (SNP) variation among FIV_Lru sequences was associated with host genetic distances and geographic location, with FIV_Lru genetic discontinuities precisely correlating with known urban barriers to host dispersal. An effect of forest land cover on FIV_Lru SNP variation was likely attributable to host population structure and differences in forest land cover between different populations. Taken together, these results suggest that the spread of FIV_Lru is constrained by large-scale urban barriers to host movement. Although urbanisation at fine spatial scales did not appear to directly influence virus transmission or spread, we found evidence that viruses transmit and spread more quickly through areas containing higher proportions of natural habitat. These multiple lines of evidence demonstrate how urbanisation can change patterns of contact-dependent pathogen transmission and provide insights into how continued urban development may influence the incidence and management of wildlife disease.