The interaction between Plasmodium vivax Duffy binding protein (PvDBP) and Duffy antigen receptor for chemokines (DARC) has been described as critical for the invasion of human reticulocytes, although increasing reports of P. vivax infections in Duffy-negative individuals questions its unique role. To investigate the genetic diversity of the two main protein ligands for reticulocyte invasion, PvDBP and P. vivax Erythrocyte Binding Protein (PvEBP), we analyzed 458 isolates collected in Cambodia and Madagascar from individuals genotyped as Duffy-positive. First, we observed a high proportion of isolates with multiple copies PvEBP from Madagascar (56%) where Duffy negative and positive individuals coexist compared to Cambodia (19%) where Duffy-negative population is virtually absent. Whether the gene amplification observed is responsible for alternate invasion pathways remains to be tested. Second, we found that the PvEBP gene was less diverse than PvDBP gene (12 vs. 33 alleles) but provided evidence for an excess of nonsynonymous mutations with the complete absence of synonymous mutations. This finding reveals that PvEBP is under strong diversifying selection, and confirms the importance of this protein ligand in the invasion process of the human reticulocytes and as a target of acquired immunity. These observations highlight how genomic changes in parasite ligands improve the fitness of P. vivax isolates in the face of immune pressure and receptor polymorphisms. Author summary Until recently, P. vivax was thought to infect only Duffy positive individuals, due to its dependence on binding the Duffy blood group antigen as a receptor for reticulocyte invasion and to be absent from parts of Africa where the Duffy-negative phenotype is highly frequent. However, a number of recent studies from across sub-Saharan Africa have reported P. vivax infections in Duffy-negative individuals. Invasion into Duffy-positive reticulocytes is mediated by the P. vivax Duffy binding protein (PvDBP). The mechanism for invasion into Duffy-negative reticulocytes is not known. A homologue of PvDBP, namely, P. vivax erythrocyte binding protein (PvEBP), has been recently identified but its role in Duffy independent invasion is not clearly defined. Here, we provide unique insights into the roles of these two key ligands by studying the genetic diversity of P. vivax isolates collected from Cambodia, where most of the individuals are Duffy positive (not all), and Madagascar where both Duffy-positive and Duffy-negative individuals coexists. Our data suggest that PvEBP may play an important functional role in invasion into Duffy-negative reticulocytes. PvEBP appears to be a target of naturally acquired antibody responses following natural exposure to P. vivax infection and such as a consequence an important vaccine candidate, together with PvDBP.