Aims: To achieve reliable detection of methicillin resistance in clinical isolates of coagulase-negative staphylococci. Methods and Results: Strains (105) were evaluated by normatized antimicrobial susceptibility methods, and for the presence of the methicillin resistance-determining mecA gene, using the polymerase chain reaction. Correlation between phenotypic and genotypic methods was obtained in 87.6% of the samples. Six strains, classified as methicillin-susceptible by phenotypic assays, revealed the presence of the mecA gene, indicating that methicillin, resistance expression was probably repressed. Another seven isolates failed to show mecA amplification after displaying methicillin resistance in phenotypic evaluations. The susceptibility of the methicillin-resistant isolates to other antimicrobial agents was variable. Conclusions: Genotypic determination of the mecA gene proved to be the most reliable method for detection of methicillin resistance. Significance and Impact of the Study: Correct assessment of methicillin resistance, such as that attained through genotyping, is essential for defining therapeutic strategies, particularly when treating severely compromised patients.