Merozoite surface protein 1 (MSP-1) is a polymorphic malaria protein with functional domains involved in parasite erythrocyte interaction. Plasmodium vivax MSP-1 has a fragment (Pv200L) that has been identified as a potential subunit vaccine because it is highly immunogenic and induces partial protection against infectious parasite challenge in vaccinated monkeys. To determine the extent of genetic polymorphism and its effect on the translated protein, we sequenced the Pv200L coding region from isolates of 26 P. vivax-infected patients in a malaria-endemic area of Colombia. The extent of nucleotide diversity (π) in these isolates (0.061 ± 0.004) was significantly lower (P ≤ 0.001) than that observed in Thai and Brazilian isolates; 0.083 ± 0.006 and 0.090 ± 0.006, respectively. We found two new alleles and several previously unidentified dimorphic substitutions and significant size polymorphism. The presence of highly conserved blocks in this fragment has important implications for the development of Pv200L as a subunit vaccine candidate.