MRSA CC398 is a clone of S. aureus that has recently emerged in pigs and other domestic animals worldwide. As any other MRSA, the clone displays high levels of antibiotic resistance and poses a serious threat to human health because of the risk of antibiotic treatment failure in human patients. We developed a new diagnostic test for identification of MRSA CC398 using a single one-step PCR that is very easily performed within a few hours. The test is based on the principle that clonal differences within S. aureus are reflected in the sequence of a gene (sau1hsdS1) located on the chromosome of this bacterial species. Accordingly, such a gene represents an optimal target for S. aureus and MRSA identification at the clone level. The test includes detection of the gene conferring methicillin resistance (mecA), therefore allowing rapid discrimination between methicillin-susceptible and methicillin-resistant variants of the clone. A preliminary validation of the test was performed on a collection of CC398 and non-CC398 strains, resulting in 100% sensitivity and 100% specificity. The test can be combined to real-time PCR technology to further reduce simplify the test performance as well as to allow quantification of the target MRSA clone in biological specimens. The invention has important applications related to surveillance and control of MRSA CC398 in humans, animals and food products.