Large mammals have been colonising urban areas throughout the world. This process is often accompanied by genetic and behavioural changes, and as a result, urban populations may form distinct entities within continuous range of the species. In this paper, we present the results of an analysis of the spatial distribution of genetic variation in urban/suburban populations of wild boars Sus scrofa. We used a genetic variation of 12 microsatellite markers to analyse the population structure of wild boar inhabiting a large city (Kraków, Poland) and its rural surroundings. We discovered a profound differentiation between urban and rural areas, with urban individuals forming a distinct genetic group within an otherwise more continuous range of the species. The genetic distinctiveness of the urban wild boar population seems to be maintained not only by physical barriers but plausibly by behavioural differences. Although the chronology of the highway bypass construction may partly explain some of the genetic relatedness between wild boar populations, our results suggest attitudes towards humans may be an important factor influencing immigration to the areas of increased human presence. We discuss possible implications for the management of the wild boar in the city.