In the European Union, African swine fever (ASF) affects wild boar (Sus scrofa) populations in several Member States. Knowledge of population connectivity is important for the implementation of control measures, in particular the establishment of effective barriers. Population genetic comparisons of neighbouring populations can be very helpful in this respect. The present study investigated the genetic differentiation of wild boar in eastern Germany. This region has been affected by ASF since September 2020. A total of 1,262 wild boars from 31 hunting grounds (populations) in ASF-affected and ASF-free districts were sampled over a total area of almost 100,000 km². The study area encompassed a network of geographical factors that promote (roads, rivers, cities) or inhibit (natural areas, habitat corridors) genetic differentiation between wild boar populations. The genetic differentiation of the areas was based on 12 microsatellite markers. Three different Bayesian algorithms were used to analyse the data. The results were combined into a common approach with 9 clusters. Based on the cluster distribution in each population, the connectivity between the areas was quantified. The strongest differentiation was found along an imaginary line along the lower Elbe valley through Berlin and the A11 freeway to the Szczecin Lagoon. In contrast, the Mecklenburg Lake District and the south-east of the study area showed strong connectivity between areas. The special features of the landscapes along the lower Elbe valley, which was assessed as highly connective, and the high barrier effect of the A11 freeway in contrast to the other freeways in the study area show that barrier effects cannot be generalised in principle, but are actually determined by the circumstances of individual structures. The results of the connectivity analysis were compared with the distribution of viral lineages and variants. The genotypes of the wild boar populations and the ASFV lineages and variants showed a good explanatory approach for the observed disease dynamics in the study area. The newly gained knowledge on barriers and regionally different connectivity between wild boar populations can support considerations and measures for the containment of ASF in the affected areas by improving the understanding of wild boar dispersal dynamics.