Coagulase-negative staphylococci (CoNS) are among the main causative agents of nosocomial infections, and multi-resistant strains has become a major issue over the past decades. The objectives of this study are to determine the frequency of oxacillin resistance among CoNS strains isolated from blood cultures of patients admitted at the University Hospital of the Faculty of Medicine of Botucatu, Brazil, and to compare phenotypic methods with the gold standard (amplification of the mecA gene) used for the detection of methicillin resistant-CoNS. Among the 103 CoNS strains analyzed, 85 were identified as S. epidermidis, ten as S. warneri, five as S. haemolyticus, one as S. capitis, one as S. hominis and one as S. lugdunensis. Amplification of the mecA gene revealed 85 (82.5%) positive strains. Of these, 81 (95.3%) were resistant by the oxacillin and cefoxitin disk diffusion method, 82 (96.5%) by screening on Mueller-Hinton agar supplemented with 4 µg/ml oxacillin and 4% NaCl and 84 (98.8%) by the E-test. The mecA gene was detected in 82.3% of the S. epidermidis isolates and in seven of the ten S. warneri isolates. Among the phenotypic methods studied, the E-test yielded the best results when compared to the gold standard. The results showed that most of the CoNS strains were resistant to oxacillin and to multiple drugs; a fact making treatment of infections caused by these microorganisms more difficult.