Objectives: To develop and validate a new multiplex PCR strategy for subtyping SCCmec type IV methicillin-resistant Staphylococcus aureus (MRSA) strains-SCCmec IV multiplex PCR. Methods: Seven primer pairs were designed to detect the ccrB allotype 2 (internal positive control), the five polymorphic J1 regions described so far for SCCmec type IV and the new J1 region specific for EMRSA-15. Primer sets were tested for specificity and robustness with prototype strains for each subtype of SCCmec type IV. The multiplex PCR conditions were optimized in a trial-error approach. Results: The seven prototype strains for the earlier described subtypes of SCCmec type IV and the EMRSA-15 prototype strain were correctly characterized by our strategy. Moreover, 13 diverse SCCmec type IV strains could be assigned to a subtype of SCCmec type IV and 5 EMRSA-15 strains were assigned to the new subtype IVh. One strain could not be assigned to an SCCmec type IV subtype because of the absence of amplification of the specific J1 region. Conclusions: This new strategy, based on a single multiplex PCR reaction, is adequate for the rapid assignment of all major subtypes of SCCmec type IV described so far and also the new subtype IVh characteristic of EMRSA-15. This strategy complements well the previously described multiplex PCR assay for the rapid assignment of SCCmec types.