We compared real-time polymerase chain reaction (RT-PCR) with in vitro culture for detecting methicillin-resistant Staphylococcus aureus in samples from environmental surfaces. The sensitivity of RT-PCR, compared with culture, was 92.5%, and the specificity was 51.4%. Because of poor specificity, the RT-PCR kit tested is not suitable for the detection of MRSA on hospital surfaces.