Pv12, Pv38 and Pv41, the three 6-Cys family proteins which are expressed in the blood-stage of vivax malaria, might be involved in merozoite invasion activity and thus be potential vaccine candidate antigens of Plasmodium vivax . However, little information is available concerning the genetic diversity and natural selection of these three proteins. In the present study, we analyzed the amino acid sequences of P. vivax blood-stage 6-Cys family proteins in comparison with the homologue proteins of Plasmodium cynomolgi strain B using bioinformatic methods. We also investigated genetic polymorphisms and natural selection of these three genes in P. vivax populations from the China-Myanmar endemic border. The three P. vivax blood-stage 6-Cys proteins were shown to possess a signal peptide at the N-terminus, containing two s48/45 domains, and Pv12 and Pv38 have a GPI-anchor motif at the C-terminus. Then, 22, 21 and 29 haplotypes of pv12 , pv38 and pv41 were identified out of 45, 38 and 40 isolates, respectively. The dN/dS values for Domain II of pv38 and pv41 were 3.33880 and 5.99829, respectively, suggesting positive balancing selection for these regions. Meanwhile, the C-terminus of pv41 showed high nucleotide diversity, and Tajima’s D test suggested that this fragment could be under positive balancing selection. Overall, our results have significant implications, providing a genetic basis for blood-stage malaria vaccine development based on these three 6-Cys proteins. [ABSTRACT FROM AUTHOR]