Staphylocoagulase (SC) is a major phenotypic determinant of Staphylococcus aureus. Antigenic specificity of SC (SC serotype) has been classified into at least 10 types and employed as an epidemiological marker. In the present study, from the sequence information of SC genes, a novel multiplex PCR assay was developed to determine SC genotypes (SC types) I-X corresponding to SC serotypes I-X, respectively, and to discriminate two subtypes of SC types IV and V. Two PCR reactions (Sc-R1 and Sc-R2) for a single isolate were designed for assigning common SC types in S. aureus isolates from humans (I-VIII and X). When amplicon was not produced, an additional PCR (Sc-R3) was performed to assign SC type IX or subtype Vb. Subtypes IVa and IVb were discriminated by an additional PCR (Sc-R4). SC types were discriminated successfully for the S. aureus strains with established SC types I-X including two subtypes of IV and V. The multiplex PCR assay established in this study could assign SC types for S. aureus clinical isolates at a high determination rate, providing more accurate information on incidence of SC types, and was considered to be useful for epidemiologic characterization of S. aureus.