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<p>Abstract</p> <p>Background</p> <p><it>Plasmodium vivax</it> reticulocyte binding protein-2 (PvRBP-2) is a promising candidate for development of vaccine against parasite. DNA sequence polymorphism in <it>pvrbp-2</it> which may hamper the vaccine development program has been identified in laboratory strains. Therefore, unraveling genetic polymorphism in <it>pvrbp-2</it> from field isolates is a prerequisite for success in vaccine development. This study was designed with a primary aim to uncover genetic polymorphism in <it>pvrbp-2</it> among <it>P. vivax</it> field isolates.</p> <p>Results</p> <p>Using virtual restriction mapping of <it>pvrbp-2</it> sequences, two restriction enzymes (<it>Alu</it>I and <it>Apo</it>I) were selected for the development of <it>pvrbp-2</it> as a PCR-RFLP marker. Restriction fragment length polymorphism (RFLP) analysis revealed a high degree of genetic polymorphism in the <it>pvrbp-2</it> gene among field isolates of <it>P. vivax</it>. <it>Apo</it>I-RFLP was found to be more efficient in identifying the extent of genetic polymorphism in <it>pvrbp-2</it> compared to <it>Alu</it>I-RFLP. Combined genotyping/haplotyping of RFLP pattern revealed a total of 36 distinct RFLP patterns among 83 <it>P. vivax</it> isolates analyzed. DNA sequence analysis also supports high degree of genetic polymorphism among field isolates of <it>P. vivax</it>. <it>Pvrbp-2</it> PCR-RFLP method is able to distinguish multiple infection up to 16.86% and it revealed a low level of shared genetic pool between more than two populations.</p> <p>Conclusion</p> <p>The study suggests that <it>pvrbp-2</it> is highly polymorphic genetic marker which can be used for population genetic analyses. RFLP analysis suggests presence of nearly similar proportion of <it>Sal-1</it> and <it>Belem</it> alleles in Indian <it>P. vivax</it> populations. The larger extent of genetic polymorphism identified from limited samples advocates to screen genetic polymorphism in <it>pvrbp-2</it> from malaria endemic geographical regions and countries for designing <it>pvrbp-2</it> based anti-malarial control measures.</p>