Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization is crucial for the prevention and control of MRSA infections in health care settings. The LightCycler MRSA Advanced Test (Roche Diagnostics) is a commercially available real-time PCR assay for direct detection of MRSA nasal colonization by targeting of the staphylococcal cassette chromosome mec (SCCmec)-orfX junction. The diagnostic performance of the assay was compared with that of ChromID MRSA agar (bioMérieux) culture and an in-house duplex real-time PCR assay. Among 1,246 nasal swab specimens collected from 2 general hospitals in Hong Kong, 174 (14%) were considered true positive for MRSA. Chromogenic culture and the in-house real-time PCR assay identified 147 (84.5%) and 133 (76.4%) true-positive cases with specificities of 100% and 98.6%, respectively. Based on the target melting temperature (Tm) values (57.0 to 62.0 °C) defined by the manufacturer, the LightCycler MRSA Advanced Test identified only 85 (48.9%) true-positive specimens. Interestingly, an additional 60 (34.5%) true-positive specimens were detected despite atypical Tm values of 55 °C, providing overall sensitivity and specificity values of 83.3% and 99%, respectively. Among isolates with Tm values of 55 °C, most were typed as clonal complex 45 (CC45). By sequence analysis of the SCCmec-orfX junction, characteristic single-nucleotide polymorphisms (SNPs) were identified only in isolates with Tm values of 55°C and not in those with typical Tm values. It is conceivable that those SNPs were located inside the target region of the proprietary hybridization probes, which resulted in a Tm shift in the melting curve analysis. Our study highlights the importance of a global evaluation of commercial kits so that the interpretation algorithm covers different lineages of MRSA clones prevalent in various geographical regions.