SummaryTwo polymorphic marker genes, merozoite surface protein 3a (PvMSP3a) and merozoite surface protein 3b (PvMSP3b), from 100 Plasmodium vivax field isolates, were investigated using polymerase chain reaction and restriction fragment length polymorphism (PCR ⁄ RFLP). Genotyping of PvMSP3a and PvMSP3b revealed marked polymorphisms in length and sequence. Three major types of PvMSP3a (Type A, B and C) and two major types of PvMSP3b (Type A and B) were detected based on the length of PCR products. Fourteen alleles of both genes with difference frequencies were distinguished by restriction fragment length polymorphism, and these results strongly support that P. vivax isolates in Thailand are markedly diverse. PvMSP3a and PvMSP3b are reliable polymorphic markers for population genetic analysis of P. vivax, and PCR ⁄ RFLP provides a powerful method for genotyping and identification of mixed parasite infections without requirement of gene sequencing.keywords Plasmodium vivax, genotyping, polymorphic marker genes, merozoite surface protein 3a, merozoite surface protein 3b