A multiplex polymerase chain reaction (PCR) assay using oligonucleotide primers to detect mecA and 16S ribosomal RNA gene was developed to aid in identification of methicillin-resistant staphylococci. Validation included 99 isolates of staphylococcus grouped into one of five categories: methicillin-susceptible coagulase-negative staphylococcus (MSCNS), methicillin-resistant coagulase-negative staphylococcus (MRCNS), methicillin-susceptible Staphylococcus aureus (MSSA), high beta-lactamase producing S aureus (HiBSA), and methicillin-resistant S aureus (MRSA). mecA was detected in MRSA (21/21), and in MRCNS (20/20), but not in MSSA (0/20). mecA was occasionally detected in HiBSA (1/19) and MSCNS (3/19). This multiplex PCR assay was also used to test 30 clinical isolates of coagulase-negative staphylococci with discrepancies between results of in vitro tests for susceptibility to oxacillin and was found to be valuable when a more definitive determination of intrinsic methicillin-resistance was desired.