Staphylococcus aureus is a leading cause of bovine, ovine and caprine mastitis. We know that S. aureus demonstrates host-specificity as strains infecting a particular host are non-randomly distributed across clonal lineages. However, our understanding of how some lineages have evolved to become specialized pathogens of ruminants is very limited. Previous population studies have identified a common clonal complex CC133 which successfully infects multiple ruminant species including goats, sheep and cows but has not been isolated from humans. In order to investigate the evolutionary basis for the ruminant host-tropism of CC133, we determined the 2.85Mb genome sequence of a representative ovine mastitis isolate. Analysis of the genome revealed several novel mobile genetic elements including 3 prophages and 2 variants of the Staphylococcal pathogenicity island family which have not been identified among human S. aureus isolates. Several novel putative virulence loci were identified which may contribute to host-specificity including a phage-encoded superantigen, a von willebrand factor-binding protein and a new member of the SDR family of MSCRAMMs. Comparative genomic hybridisations of a range of diverse strains of bovine, ovine and caprine origin using a microarray specific for the CC133 ovine strain, 7 sequenced human strains and bovine strain RF122 have allowed identification of the gene complement required for S. aureus ruminant mastitis. Taken together, these data provide many new avenues for future studies into the molecular basis of intra-mammary infection and the rational design of new therapeutics.