This study aimed to identify the genetic variation and distribution of malaria vector mosquitoes to provide information on vector introduction and movement in the Republic of Korea (ROK).Anopheles sinensis and Anopheles kleini, presumed to be competent malaria vectors in ROK, were analyzed using the cytochrome c oxidase subunit I (COI) gene marker.Both species exhibited sequence characteristics of migratory populations. Some dominant haplotypes in this study were identical to reference sequences from China and Russia. This confirmed that noticeable genetic differences could arise due to distance and terrain. In addition, the variation within population had substantial impact on the overall variation.This study provides an understanding of the unique characteristics of malaria vectors in ROK. Continuous research on the population genetic structure will enhance the molecular biological understanding of vector populations and can serve as foundation data for identifying methods for malaria eradication.