The MSX2 gene encodes a homeodomain transcription factor important for normal head and face morphogenesis. MSX2 is expressed in key craniofacial structures during development and mutations in the human gene give rise to various craniofacial abnormalities. We are interested in the genetic basis of non-pathogenic variation in skull and face shape. As part of this study we have analysed DNA from a panel of different dog breeds, selected for the differences they show in these traits and investigated MSX2 as a candidate gene. In this paper we describe the cloning of the canine homologue of MSX2, the determination of its structure, sequence and localization of the gene to dog chromosome 4q23. The DNAs from 11 individual domestic dogs belonging to 10 different breeds were sequenced in a search for genetic variation. Our studies show that variation in MSX2 does not contribute to the diversity of face shape observed in these domestic dogs and that the MSX2 sequence is strongly conserved between different dog breeds. The proximal promoter shows a high level of interspecies sequence conservation and several conserved transcription factor binding motifs have been identified and their significance discussed.