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Background:Malaria is one of the major communicable diseases in India and worldwide. PvMSP3β is a highly polymorphic gene due to its large insertions and deletions in the central alanine-rich region, which, in turn, makes it a valuable marker for population genetic analysis. Very few studies are available from India about the genetic diversity of Plasmodium vivax based on PvMSP3β gene, and hence, this study was designed to understand the molecular diversity of the P. vivax malaria parasite. The accumulating epidemiological data provide insights into the circulating genetic variants of P. vivax in India, and ultimately benefits the vaccine development.Materials and Methods:A total of 268 samples confirmed to be positive by microscopy, rapid diagnostic test, and quantitative buffy coat test were collected from four different regions of India (Puducherry, Mangaluru, Jodhpur, and Cuttack) in the present study. Polymerase chain reaction (PCR)-based diagnosis was carried out to confirm the P. vivax monoinfection, and only the mono-infected samples were subjected to PvMSP3β gene amplification and further restriction fragment length polymorphism (RFLP) to determine suballeles.Results:Based on the size of the amplified fragment, the PvMSP3β gene was apportioned into two major types, namely Type A genotype (1.6–2 Kb) was predominantly present in 148 isolates and Type B (1–1.5 Kb) was observed in 110 isolates. The percentage of mixed infections by PCR was 3.73%. All the PCR products were subjected to RFLP to categorize into suballeles and we detected 39 suballeles (A1–A39) in Type A, and 23 suballeles (B1–B23) in Type B genotype. A high degree of diversity was observed among the isolates collected from Mangaluru region when compared to isolates collected from other regions.Conclusion:The present study showed a high degree of genetic diversity of PvMSP3β gene among the isolates collected from various parts of India. High polymorphism in PvMSP3β gene makes it a promising marker for epidemiological and vaccine development studies.