A freshwater mussel,Nodularia breviconcha(Mollusca: Bivalvia: Unionida) is endemic to Korean Peninsula. It has recently been taxonomically reexamined and elevated from a subspecies ofN.douglasiaeto an independent species. But population genetic studies for the species have rarely been conducted. To explore the population genetic structure ofN.breviconcha, the nucleotide sequences of cytochrome oxidase subunit I(COI) and 16S rRNA genes from 135N.breviconchaindividuals, including 52 from this study and 83 from Choi et al. (2020). We found 23 COI and 11 16S rRNA genes haplotypes. Phylogeny, TCS network, Principal coordinates analysis, and spatial analysis of molecular variance performed with COI gene indicated that there are exist three different genetic lineages in theN.breviconchapopulations: West lineage, Southwest lineage, and Southeast lineage. According to the time calibrated phylogeny, they are likely to be diverged during the late Miocene (8–6 Ma). Geographical distribution patterns of the three genetic lineages may be related to the formation of Taebaek and Sobaek-Noryeong mountain ranges in the Korean Peninsula occurred during the Miocene (30–10 Ma). The present results of this study will be helpful not only for the conservation, but also for the exploration of the population genetic structure of endemic freshwater mussels in the Korean Peninsula.