Approximately 90 million people are suffered from vivax malaria annually. This disease imposes a heavy economic cost in endemic areas of Asia and the Americas. This study was design and implemented to reveal the extent of polymorphism of the Plasmodium vivax Merozoite Surface Protein -3a gene. 37 blood samples (20 Iranian and 17 non-Iranian) collected from patients attending to malaria clinics in the Sistan and Baluchistan province. The DNA was amplified by nested PCR and the products were digested with HhaI enzyme through PCR/RFLP technique. Three biotypes of the gene based on the size of PCR products, including A (about 1900bp), B (about 1400bp) and C (about 1100bp) and 10 allelic groups after digesting of PCR products with HhaI enzyme was observed. A significant difference between the two groups of patients was not detected.We observed that P. vivax isolates of Sistan and Bluchestan were extremely diverse, and the results are almost compatible with the results of other studies performed previously. Based on the results of our study RFLP method using HhaI (small fragments from 50-500bp) is suitable technique for describing diversity of the gene.