The region II of Plasmodium vivax Duffy binding protein (PvDBP-II) contains the critical binding residues, which is a major target for development of naturally acquired immunity. Several studies showed sequence polymorphisms in PvDBP-II, which may inhibit antibodies recognition. Therefore, in this study the level of PvDBP-II polymorphism within and among P. vivax populations from re-emerged areas in north and endemic areas in south of Iran were evaluated by sequencing analysis in 75 isolates for the first time. Fourteen non-synonymous and one synonymous mutations were identified and none of the amino acid substitutions were directly involved in erythrocyte binding. Only 6 out of 14 detected mutations have been found among northern isolates, including D384G, R390H, N417K, L424I, W437R, and I503K. In total, two and nine different variants have been identified among northern and southern isolates, respectively. High association of the amino acid frequencies for codons 417, 437, and 503 were found among northern (85% for trio association and 100% for N417K with W437R), and southern (36% for trio association and 98% for N417K with W437R) samples. Polymorphisms at positions R308S, K371E, D384G, K386N, R390H, N417K, L424I, W437R, and I503K were identified from Iran and diverse geographic areas; however, mutation at position F306L was only reported from Asian malaria endemic areas. It is suggested that to develop polyvalent vaccine against P. vivax infection, it is better to incorporate the common and high prevalent allelic variants of the antigen that were reported from different malaria endemic regions.