Genetic variation among populations of commensal house mice was studied across the territories of the Czech and Slovak Republics and in some adjacent areas of Germany. We used six diagnostic allozyme loci (Es-2, Gpd-1, Idh-1, Mpi, Np, Sod-1) and the following molecular markers: B1 insertion in the Btk gene (X chromosome), Zfy2 18-bp deletion (Y chromosome), BamH I restriction site in the mt-Nd1 gene (mtDNA) and Hba-4ps 16-bp insertion (diagnosing the presence of t haplotypes). In total, 544 individuals taken from 49 localities were examined. Almost the entire territories of the Czech Republic and Slovakia were found to be occupied by Mus musculus, the only exception being the westernmost parts of the Czech Republic, where M. musculus meets the range of M. domesticus and forms a narrow belt of hybrid populations. Despite this, domesticus-type alleles of some allozyme markers (notably Es-2) were also found at sites well within the range of M. musculus, either tens or hundreds of kilometres behind the hybrid zone. This provides evidence of either: (1) introgression of some markers into the species’ genome due to free gene flow through the zone, or (2) human-mediated long-distance migrations, or (3) incomplete lineage sorting. Conversely, variants of molecular markers typical for M. domesticus in Btk, Zfy2 and mt-Nd1were only found in the westernmost populations studied. t haplotypes were quite frequent in some populations, irrespective of whether M. domesticus, M. musculus or their hybrids, yet no t/t homozygotes were found. The mean frequency of t/+ heterozygotes found within the study populations was 13%.