Especially in urbanized landscapes, habitat fragmentation and increasing numbers of infrastructural features may limit genetic exchange among wildlife populations. Yet, whether this results in genetic differentiation among individuals in different habitat fragments will depend on both the species studied and the composition of the landscape. European pine martens (Martes martes) show clear spatial structure at a Europe-wide scale, but whether gene flow among habitat patches can be maintained at a more local scale in intensively urbanized areas remained unclear. Here, we analysed genotypic data from 270 pine martens sampled from locations scattered across the Netherlands, one of the most densely populated areas in the world. Using Bayesian clustering models we show that most likely maximum two large subpopulations exist in the Netherlands. We observed relatively low levels of genetic differentiation and genetic evidence of regular long-distance dispersal by juveniles that must have crossed one or multiple major highways. Our results suggest that genetic exchange among Dutch pine martens has, until 2010, not been impacted severely by the countries’ dense infrastructural network. Furthermore this species seems to have maintained its genetic diversity despite a recent demographic bottleneck. These conclusions support the idea that the effects of habitat fragmentation may strongly differ between (groups of) species, and that prioritization and optimization of management decisions thus requires direct study of the targeted species.