Staphylococcus haemolyticus is an opportunistic bacterial pathogen that colonizes human skin and is remarkable for its highly antibiotic-resistant phenotype. We determined the complete genome sequence of S. haemolyticus to better understand its pathogenicity and evolutionary relatedness to the other staphylococcal species. A large proportion of the open reading frames in the genomes of S. haemolyticus, Staphylococcus aureus, and Staphylococcus epidermidis were conserved in their sequence and order on the chromosome. We identified a region of the bacterial chromosome just downstream of the origin of replication that showed little homology among the species but was conserved among strains within a species. This novel region, designated the "oriC environ," likely contributes to the evolution and differentiation of the staphylococcal species, since it was enriched for species-specific nonessential genes that contribute to the biological features of each staphylococcal species. A comparative analysis of the genomes of S. haemolyticus, S. aureus, and S. epidermidis elucidated differences in their biological and genetic characteristics and pathogenic potentials. We identified as many as 82 insertion sequences in the S. haemolyticus chromosome that probably mediated frequent genomic rearrangements, resulting in phenotypic diversification of the strain. Such rearrangements could have brought genomic plasticity to this species and contributed to its acquisition of antibiotic resistance.As a part of the normal bacterial flora, staphylococci colonize the skin and mucosal membranes of humans. In addition, staphylococci frequently cause opportunistic infections in patients with underlying disease, such as those with prosthetic devices, surgical patients, individuals undergoing dialysis, or patients with diabetes. Since antibiotic chemotherapy was introduced in the last century, staphylococci have successfully persisted by altering their genetic traits to avoid being killed. Multidrug-resistant staphylococcal strains, exemplified by methicillin-resistant staphylococci, are now prevalent worldwide. Among 40 staphylococcal species described to date, Staphylococcus aureus is the most virulent species and poses the greatest threat in hospitals worldwide. In addition to its nosocomial spread, S. aureus has also become problematic in community settings, where individuals without predisposing factors have acquired methicillin-resistant staphylococcal infections. Although most community-acquired staphylococcal infections involve the skin and soft tissues, some otherwise healthy children have acquired potentially lethal S. aureus infections with severe symptoms, such as necrotizing pneumonia (5,28,29).To gain a better understanding of the overall pathogenesis of staphylococcal infections and identify novel targets for new chemotherapeutic agents, researchers have sequenced the genomes of seven S. aureus strains (3,9,13,22). A comparative analysis of these strains has revealed that many genes involved in staphylococcal pathogenicity and...