The aim of this study was to evaluate retrospectively the performance of the Xpert MRSA assay in routine practice and its current use in the intensive care unit (ICU) setting of our hospital, since a pre-emptive isolation strategy has been applied. A total of 6473 patients were routinely screened with ESwab for methicillin-resistant Staphylococcus aureus (MRSA) using three generations of rapid real-time polymerase chain reaction (PCR) (Cepheid GeneXpert) over three consecutive periods of time. Performance was evaluated using broth enrichment culture as the reference method. Our results show that the last generation of Xpert MRSA (NxG) assay is more specific (99.2% vs. 97.9%) but not more sensitive (77.8% vs. 86.9%) than the third generation. Considering the low prevalence of MRSA in our hospital, we obtained an overall low positive predictive value. In conclusion, it remains difficult to abandon the reference method in routine practice considering the possible implications of an erroneous MRSA result in the ICU.