We analyzed the genetic structure and relationships of house mouse (Mus musculus) populations in the remote Atlantic archipelago of the Azores using nuclear sequences and microsatellites. We typed Btk and Zfy2 to confirm that the subspecies Mus musculus domesticuswas the predominant genome in the archipelago. Nineteen microsatellite loci (one per autosome) were typed in a total of 380 individuals from all nine Azorean islands, the neighbouring Madeiran archipelago (Madeira and Porto Santo islands), and mainland Portugal. Levels of heterozygosity were high on the islands, arguing against population bottlenecking. The Azorean house mouse populations were differentiated from the Portuguese and Madeiran populations and no evidence of recent migration between the three was obtained. Within the Azores, the Eastern, Western, and Central island groups tended to act as separate genetic units for house mice, with some exceptions. In particular, there was evidence of recent migration events among islands of the Central island group, whose populations were relatively undifferentiated. Santa Maria had genetically distinctive mice, which may relate to its colonization history.