Aminoglycosides are potent bactericidal agents that play an important role in antistapylococcal therapy. In this study, we used a multiplex polymerase chain reaction assay to investigate the prevalence of aac(6')-Ie/aph(2''), ant(4')-Ia, and aph(3')-IIIa, the genes encoding the most clinically prevalent aminoglycoside-modifying enzymes, and simultaneous detection of the methicillin resistance gene, mecA, in Staphylococcus aureus isolates. A total of 100 S. aureus clinical isolates were collected and tested by disk diffusion and agar dilution method for susceptibility testing. All isolates were screened for the presence of the three aminoglycoside-modifying enzyme genes and the methicillin resistance gene. The ant(4')-Ia was the most frequent gene (58%), and aac(6')-Ie/aph(2'') and aph(3')-IIIa genes were found in 46% and 6% of the isolates, respectively. All isolates harboring the aac(6')-Ie/aph(2'') gene were resistant to gentamicin (100% concordance). Seventy-one percent of the isolates demonstrated resistance to at least one of the aminoglycosides tested. PCR results showed that 53% of all isolates harbored the mecA gene. Aminoglycoside resistance was closely associated with oxacillin resistance (p < 0.05). In conclusion, because of the high rate of aminoglycoside resistance among S. aureus clinical isolates observed in this study, periodic surveillance on the resistance prevalence should be performed.