Background <p><i>Anopheles kleini</i> is a competent vector mainly observed in the northern, malaria-risk areas of the Republic of Korea (ROK). In this study, the population genetic structure of <i>An. kleini</i> was analysed for the first time in the ROK using the mitochondrial cytochrome <i>c</i> oxidase subunit I (<i>COI</i>) marker.</p> Methods <p>The genetic structure of 249 <i>An. kleini</i> was analysed from three statistically analysable regions, each including more than five mosquitoes.</p> Results <p>Network analysis identified 140 haplotypes organized into three clusters. Cluster II was related to <i>An. kleini</i> from eastern Russia and northwestern China. The pairwise genetic distance (<i>F</i>_ST) values among the populations showed regional genetic differences between Gangwon-do and Gyeonggi-do. Analysis of molecular variance (AMOVA) indicated that individual mosquitoes within the population had a significant influence on the total variation. The neutrality test, using three methods (Fu’s Fs, Fu, and Li’s D, and Fu and Li’s F), indicated that all values were negative, suggesting that <i>An. kleini</i> is an expanding population. <i>Anopheles kleini</i> in Yanggu has a significant difference in genetic distance from other regions.</p> Conclusion <p>This study provides molecular epidemiologically information for understanding the spatial population structure of <i>An. kleini</i> and is helpful for malaria control in the ROK.</p>