Staphylococcus aureus is recognized worldwide as a major bovine mastitis pathogen causing marked financial losses to the dairy industry. Chronically infected cows are the main reservoir of the infection in a herd. However, the actual role of asymptomatic mammary glands as carriers for pathogenic microorganisms has never been described. Accordingly, the aim of this study was to evaluate the diversity of S. aureus isolated from these bovine mammary glands, previously identified by the absence of clinical signs of inflammation or detectable alterations in the milk secretion. In a single dairy herd, mammary glands from eighty-three cross-breed cows were examined. S. aureus was isolated from milk samples of individual quarters from animals showing negative California Mastitis Test (CMT) reaction and quarter somatic cells count (QSCC) < 200.000 cells/mL. Each morning, from these same mammary glands, during a 16 day-follow up period, milk samples were collected, and after CMT testing were submitted to microbiological examination and QSCC evaluation. Thirty-one S. aureus isolates were isolated and identified by morphology and biochemical tests. Macrorestrictions of chromossomal DNA of the isolates were performed by PFGE using SmaI endonuclease. Genotyping of the 31 isolates revealed just a single pulsotype. Also, the same pulsotype was observed in the same mammary gland for a period of 16 days. In conclusion, the genetic similarity presented by S. aureus isolates during the observed period from the same mammary glands demonstrated the carrier status.