Blood cultures positive for gram-positive cocci in clusters can pose a dilemma for empiric antimicrobial therapy because they could represent coagulase-negative staphylococcus or Staphylococcus aureus bacteremia. The GeneXpert MRSA/SA BC Assay (Cepheid, Sunnyvale, CA) is a polymerase chain reaction-based method for identifying S aureus and methicillin resistance that has been approved for use in adults, but data on its use in samples from pediatric patients is limited. We validated the Xpert MRSA/SA BC Assay for use with anaerobic and polymicrobial specimens from pediatric patients and implemented it for routine presumptive identification of S aureus in our pediatric hospital. The assay was 100% sensitive and specific for methicillin-resistant S aureus and 100% sensitive and 99.5% specific for methicillin-susceptible S aureus. Time to presumptive identification of S aureus bacteremia and determination of methicillin susceptibility was reduced by more than 24 hours. We found the Xpert MRSA/SA BC Assay to be a rapid, accurate tool for detecting methicillin-resistant and methicillin-susceptible S aureus in positive pediatric blood cultures, including polymicrobial cultures and those recovered in anaerobic blood culture media.