Multiresistant staphylococci (82 Staphylococcus aureus and 114 coagulase-negative staphylococci) were characterized by testing with rapid multiplex polymerase chain reaction (PCR) assays for species identification and detection of associated antibiotic resistance genes. These 196 staphylococci were isolated from 149 adult patients who developed wound infection after elective coronary artery bypass grafts and/or valve surgery. The multiplex PCR assays allowed identification of the most common staphylococcal species with S. aureus- and Staphylococcus epidermidis-specific primers as well as the detection of the erythromycin resistance genes ermA, ermB, ermC and msrA, the aminoglycoside resistance gene aac(6')-aph(2"), the oxacillin resistance gene mecA and the penicillin resistance gene blaZ. There was a very good correlation between the genotypic analysis by PCR and the phenotype determined by standard methods of susceptibility testing and identification of staphylococcal species: 100% for erythromycin resistance, 98.0% for gentamicin resistance, 99.0% for oxacillin resistance, 100% for penicillin resistance and 100% for S. aureus and S. epidermidis species identification. This study suggests that the incidence and distribution of the tested clinically relevant antibiotic resistance genes in staphylococci associated with infections after cardiac surgery do not differ from those in strains from other infections. These multiplex PCR assays may be used as diagnostic tools to replace or complement standard methods of susceptibility testing and identification of staphylococci.