<p>Amphibian populations are rapidly declining worldwide due to habitat degradation and climate change, with species occupying narrow ecological niches facing particularly high extinction risks. Effective conservation requires a clear understanding of species-specific genetic diversity and population structure. In this study, we performed a population-level genetic analysis of the gold-spotted pond frog, <i>Pelophylax chosenicus</i>, a vulnerable amphibian species native to Korea and northeastern China. Microsatellite-based analyses revealed pronounced genetic structuring across populations, with fluvial systems playing a key role in delineating population boundaries. The degree of genetic differentiation was significantly related to the geographic distance between populations. Marginal populations exhibited relatively lower genetic diversity but higher pairwise genetic distances, indicating limited gene flow and strong genetic drift. Mitochondrial DNA analysis revealed the presence of two divergent phylogenetic lineages of <i>P. chosenicus</i> occupying overlapping geographic regions. Mito-nuclear discordance revealed the historical independent mitochondrial introgression into <i>P. chosenicus</i> and <i>P. plancyi</i>. Our findings offer valuable insight for the conservation of <i>P. chosenicus</i> and provide a framework for broader evolutionary studies across <i>Pelophylax</i> species.</p>