Landscape fragmentation caused by road infrastructures represents a major threat to the genetic diversity of a region. The resulting genetic isolation between subpopulations may lead to consanguinity, and consequently to population collapse and extinction. However, the construction of wildlife crossings can help maintain connectivity. In the present paper, we evaluated the genetic spatial structuring of populations of wild boars (Sus scrofa) in three areas of the Geneva region connected by an ecological corridor. Those areas are cut off either by a highway that is crossed by a wildlife overpass or by an anthropized sector. Genetic profiling with 9 nuclear microsatellite markers yielded 61 single profiles, which allowed for clustering, parentage, and linkage disequilibrium analyses, uncovering the populations’ genetic structure. We also evaluated whether the genetic structure was affected by the sex of individuals. In our analyses, all individuals clustered into a single genetic group, suggesting that no structure limited significantly the gene flow in the region. However, a recent admixture indicated a potential increase in the gene flow between two of the subpopulations due to the wildlife overpass, while the other part of the ecological corridor was not or was only partially functional. Genetic distances between males were significantly higher than between females, although the role of sex remains unclear as to its influence on population genetics. Finally, in order to avoid a subregion becoming fully isolated, urbanization planning should consider this genetic evaluation and proceed with further monitoring, especially by focusing on species more sensitive to landscape fragmentation.