BackgroundStreptococcus suisis a zoonotic pathogen that causes substantial economic losses in the pig industry and contributes to human infections worldwide, especially in Southeast Asia. Recently, a multiplex polymerase chain reaction (PCR) process was developed to distinguish disease-associated and non-disease-associated pathotypes ofS. suisEuropean strains. Herein, we evaluated the ability of this multiplex PCR approach to distinguish pathotypes ofS. suisin Thailand.ResultsThis study was conducted on 278 humanS. suisisolates and 173 clinically healthy pigS. suisisolates. PCR identified 99.3% of disease-associated strains in the human isolates and 11.6% of non-disease-associated strains in the clinically healthy pig isolates. Of the clinically healthy pigS. suisisolates, 71.1% were classified as disease-associated. We also detected undetermined pathotype forms in humans (0.7%) and pigs (17.3%). The PCR assay classified the disease-associated isolates into four types. Statistical analysis revealed that humanS. suisclonal complex (CC) 1 isolates were significantly associated with the disease-associated type I, whereas CC104 and CC25 were significantly associated with the disease-associated type IV.ConclusionMultiplex PCR cannot differentiate non-disease-associated from disease-associated isolates in Thai clinically healthy pigS. suisstrains, although the method works well for humanS. suisstrains. This assay should be applied to pigS. suisstrains with caution. It is highly important that multiplex PCR be validated using more diverseS. suisstrains from different geographic areas and origins of isolation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02888-9.