Rapid and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers is crucial for the effective control of MRSA transmission in healthcare facilities. The aim of this study was to verify the performance of the IDI-MRSA real-time PCR assay for direct MRSA detection in diverse mucocutaneous swabs from hospitalized patients. Swabs from nares (n = 522) and skin or other superficial sites (n = 478) were prospectively collected for MRSA screening from 466 patients admitted to an 858-bed teaching hospital. Swabs were inoculated onto selective chromogenic MRSA-ID agar, buffer extraction solution for IDI-MRSA assay, and enrichment broth. MRSA was detected by culture in 100 specimens from 47 patients. Compared to enrichment culture, the sensitivity and specificity of the PCR assay were 81.0 and 97.0%, respectively, and its positive and negative predictive values were 75.0 and 97.9%, respectively. The IDI-MRSA assay was more sensitive on swabs from nares (90.6%) than from other body sites (76.5%, P < 0.01). The PCR assay detected MRSA in 42 of 47 patients with culture positive study samples. Of 26 patients with culture-negative but PCR-positive study samples, 11 were probable true MRSA carriers based on patient history and/or positive culture on a new sample. The median turnaround time for PCR results was 19 h versus 3 days for agar culture results and 6 days for enrichment culture results. These data confirm the value of IDI-MRSA assay for rapid screening of MRSA mucocutaneous carriage among hospitalized patients. Cost-effectiveness studies are warranted to evaluate the impact of this assay on infection control procedures in healthcare settings.