Anopheles sinensis Wiedemann sensu stricto (s.s.) is a dominant mosquito and considered a secondary malaria vector in the Republic of Korea (ROK). Despite the potential significance for malaria control, population genetics studies have been conducted using only mitochondrial DNA (mtDNA), and studies of the genetics of hybridization have never been attempted. In this study, 346 specimens from 23 localities were subject to experiments. Among them, 305 An. sinensis s.s. specimens from 20 localities were used for mtDNA analysis, and 346 specimens comprising 341 An. sinensis s.s. from 22 localities and five Anopheles kleini Rueda from one locality were examined in the microsatellite study. Neighbor-joining analysis of pairwise F_ST and R_ST based on microsatellite results showed that the populations are divided into two groups, as did the mtDNA results. However, the Bayesian analysis and factorial correspondence analysis plots showed three distinct clusters. Among the mtDNA and microsatellite results, only microsatellites represented small but positive and significant isolation-by-distance patterns. Both molecular markers show the Taebaek and Sobaek Mountain ranges as barriers between the northern and southern parts of the ROK. The newly recognized third group suggests possible introgressive hybridization of An. sinensis s.s. with closely related species. The slightly different composition of populations in each group based on different markers is probably because of different population dynamics in each group. These results imply that there is restricted gene flow of epidemiologically important malariarelated genes between the northern and southern parts of the ROK.