A two-step triplex PCR assay targeting the mecA, femA, and nuc genes was developed for the detection of methicillin resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. The triplex PCR revealed the presence of the femA and nuc genes in all the S. aureus isolates examined (n=105). Forty-four clinical isolates were mecA positive and no foodborne isolates were mecA positive. The PCR results had a 98 or 99% correlation with the results of PBP2a latex agglutination tests or oxacillin susceptibility tests, respectively.