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Abstract Background The earliest records in Britain for the western European house mouse (Mus musculus domesticus) date from the Late Bronze Age. The arrival of this commensal species in Britain is thought to be related to human transport and trade with continental Europe. In order to study this arrival, we collected a total of 16 ancient mouse mandibulae from four early British archaeological sites, ranging from the Late Bronze Age to the Roman period. Results From these, we obtained ancient mitochondrial DNA (mtDNA) house mouse sequences from eight house mice from two of the sites dating from the Late Bronze to Middle Iron Age. We also obtained five ancient mtDNA wood mouse (Apodemus spp.) sequences from all four sites. The ancient house mouse sequences found in this study were from haplogroups E (N = 6) and D (N = 2). Modern British house mouse mtDNA sequences are primarily characterised by haplogroups E and F and, much less commonly, haplogroup D. Conclusions The presence of haplogroups D and E in our samples and the dating of the archaeological sites provide evidence of an early house mouse colonisation that may relate to Late Bronze Age/Iron Age trade and/or human expansion. Our results confirm the hypothesis, based on zooarchaeological evidence and modern mtDNA predictions, that house mice, with haplogroups D and E, were established in Britain by the Iron Age and, in the case of haplogroup E, possibly as early as the Late Bronze Age.