The Eurasian badger Meles meles has a wide distribution area ranging from Japan to Ireland. In western Europe badger habitats are severely disturbed by anthropogenic factors, leading to fragmentation into subpopulations and formation of a metapopulation substructuring of once continuous panmictic populations. We have examined the genetic structure of Dutch and Danish badger populations on a relatively small scale (within countries) and a larger scale (between countries). The levels of genetic variation of populations were moderate and did not differ significantly among populations (overall HO=0.30, overall HE=0.34). Considerable genetic differentiation between the Dutch and Danish populations was found (overall FST=0.32, mean pairwise Dutch–Danish FST=0.42), indicating a large-scale substructuring of these western European badger populations. Further analysis showed that the Danish badger population can be substructured into three clusters [P(k=3)=0.99], but the Dutch populations cluster into one more or less panmictic population [P(k=1)=0.99] with little or no substructuring. The presence of migration barriers, such as roads, together with the peninsular geography of Denmark, may have led to this structuring of badger populations. In contrast, measures that improve migration and connection to other populations from neighboring countries may have prevented substructuring of the Dutch badger population.