Aim: To evaluate a new quantitative reverse transcription-PCR (qRT-PCR) assay for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA). Methods and Results: Primers for Staphylococcus-specific regions of 16S rRNA gene, spa gene and mecA gene were newly designed. RNAs extracted from broth-cultured strains were tested by qRT-PCR targeting each primer, and the bacterial counts obtained correlated well with those counted by the plating method with detection limits of 100, 101 and 102 CFU. The qRT-PCR assay targeting the 16S rRNA was 6430-fold or more sensitive than qPCR assay. All Staph. aureus strains tested were detected and none of the other Staphylococcus species and genus strains tested cross-reacted with the assay targeting the spa gene. All MRSAs tested were detected by the assay targeting the mecA gene. Clinical samples, faecal material and bronchial washout solutions were tested by our assay, and MRSAs were detected with a high sensitivity within 6 h. Conclusion: Our qRT-PCR assay targeting three new primers to the target genes is a rapid and sensitive tool for the detection of MRSA directly from clinical samples. Significance and Impact of the Study: Because of its sensitivity and rapidity, our qRT-PCR assay is considered to be a valuable tool for clinical management.