|The house mouse, Mus Musculus, was first introduced into New Zealand in significant numbers in the mid nineteenth century. Earlier research suggests that multiple introductions of the three subspecies of house mouse M. m. domesticus, M. m. musculus and M. m. castaneus occurred at this time. While M. m. domesticus is widely distributed in New Zealand with M. m. musculus somewhat less so, the only extant evidence found to date for M. m. castaneus is in hybrid mice found principally in the southern half of the South Island. In this study, the hybrid population of mice in the South Island was examined in detail using a variety of molecular techniques. The extent of the hybrid zone was first established using a simple polymerase chain reaction (PCR) technique that enabled rapid identification of mitochondrial genomes as being M. m. domesticus, castaneus or musculus in nature. In the second part of the study, four representative mice from the hybrid zone and three M. m. domesticus from north of this zone were subjected to high density (600K) single nucleotide polymorphism (SNP) analyses in order to pinpoint regions of the genomes that differed in a significant manner between the two groups. In a refinement of these analyses, regions of each genome were identified as being domesticus-, castaneus- or musculus-like, using diagnostic SNP alleles for each sub-species. Some 170 mice, principally collected in the Southern South Island, were screened using the rapid PCR technique. In the coastal regions, all mice further south than 44oS had M. m. castaneus mtDNA whereas those north of 43oS had M. m. domesticus mtDNA. Between the two, there is a ‘contact zone’ in which both subspecies were found, sometimes in the same building. The contact zone extends approximately 50km north to south and some 30km inland. Classical tests with three nuclear DNA markers confirmed earlier work, namely, that the nuclear genomes of all mice appeared to be predominantly domesticus-like. Although there is no obvious geographical, ecological or land-use features that characterise this contact zone it may be relevant that there is the distinct change to a wetter colder climate in the south and inland of the contact zone, especially in winter. The inability to identify obvious defining features is not surprising, given that much research over many years worldwide has failed to yield an explanation for distinct mouse hybrid zones. The high density SNP analyses demonstrated that the nuclear genomes of all seven representative mice were very similar and largely (approximately 95%+) domesticus-like in nature. Despite this similarity, clusters of SNPs did reveal differences throughout the genome, often just extending over a few haplotype blocks and often encompassing much less than a million basepairs (Mb). Some of these were of biological interest, for example clusters of vomeronasal receptor genes and also genes believed to be involved in maternal-fetal conflict, which are known to vary markedly between species. The diagnostic SNP analyses confirmed that no marked differences exist between the genomes of the hybrid and M. m. domesticus mice. As stated above, the genomes were predominantly domesticus-like but small regions (~1Mb) of castaneus-like and musculus-like genome were found scattered throughout all mice but, contrary to initial expectations, there was no preponderance of castaneus-like regions in the hybrid mice. The one exception to the largely identical genomic SNP patterns, were those from the mouse collected in Hamilton. This was quite bizarre, in that its genome contains large (up to 10Mb) musculus-like regions that correspond exactly to the same regions in a common laboratory strain of mouse, C57BL/6J. I conclude that if pure M. m. castaneus mice did originally reach New Zealand, extensive ‘backcrossing’ with M. m. domesticus has virtually eliminated the castaneus genome, with just a few remnants remaining that may or may not confer some selective advantage, but could just as likely represent segregation of recognition factors that give rise to assortative mating. Thus, the most obvious and consistent genetic difference between the mice remains the original observation concerning their mitochondrial genomes and these should be examined in more detail. In future research aimed at identifying potential selective advantages that have allowed the hybrid mice to exclusively populate the southern South Island factors that relate to climate should be considered. Specifically, the fact that, in humans, some mitochondrial haplotypes are believed to confer a selective advantage in colder climates immediately suggests that examining variants of key genes involved in energy metabolism such as NADH-dehydrogenase-subunit-3 ( ND3 ) in the hybrid mice could be a profitable line of future research.