Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains often carry the Panton-Valentine leukocidin (PVL) genes. However, the specific role that PVL plays in the epidemiological features and pathogenesis of CA-MRSA infections has remained undefined and controversial. Conducting a retrospective study on a natural population of MRSA clinical isolates recovered from community and hospital patients in a large Canadian health-care region during a 6-year period, we identified the coexistence of 2 USA400 (a major clonal group of CA-MRSA) sibling strains with and without PVL genes. Polymerase chain reaction and sequence analysis indicated that the PVL-carrying prophage phiSa2mw was present in PVL(+) but absent in PVL(-) USA400 isolates. These strains shared identical genotypic and phenotypic properties and similar clinical characteristics. This study provides direct evidence that PVL genes are not necessarily the key determinants associated with the increasing dissemination of CA-MRSA strains, suggesting that the genomic milieu may play a greater role in this regard.