Multilocus sequence typing (MLST) is considered a reliable method for providing insight into the Streptococcus suis population structure, clonal relationships and the potential of particular clones to cause disease. Indeed, MLST has revealed the presence of several clonal complexes (CCs) within the Streptococcus suis population. However, the method is costly, time-consuming and difficult to use for screening large numbers of isolates. In this study, a multiplex PCR assay was developed to identify Streptococcus suis CCs that are relevant to human infections. The multiplex PCR assay was capable of simultaneously distinguishing CC1, CC25, CC28, CC104, CC221/234 and CC233/379, which are related to human infections in Thailand, in a single reaction. The multiplex PCR assay is useful for low-cost screening of large numbers of isolates with rapid analytical capacity and could be utilized in most laboratories.