A multiplex PCR assay for differentiation of Streptococcus parauberis serotypes was developed. The three primer pairs for subserotypes Ia and Ib/Ic and serotype II were designed from the serotype-specific sequences of the wzy gene in the loci for capsular polysaccharide biosynthesis. All of 188 S. parauberis isolates from Japanese flounder showed positive reaction with the expected size of PCR product for each serotype, which was consistent with the results of agglutination test using rabbit antisera. The nontypeable isolates, which are not differentiated by agglutination test, could be identified as subserotype Ib/Ic or serotype II. Other streptococci including S. parauberis derived from cow and major bacterial fish pathogens showed negative reaction.