A multiplex PCR was developed to detect 15 serotypes of Streptococcus suis. This multiplex PCR was separated into two reaction sets. The first set identified nine serotypes (serotypes 1/2, 1, 2, 3, 4, 7, 9, 14 and 16) and the second set identified six serotypes (serotypes 5, 8, 10, 19, 23 and 25). This assay correctly detected serotypes 2, 5 and 14 in human isolates, and serotypes 1, 2, 1/2, 3, 4, 5, 7, 9, 14, 16 and 19 in pig isolates from Thailand. No cross-reaction was observed with other streptococcal species. This assay may be useful for the serotype surveillance of human and pig isolates of S. suis.