Coldwater‐adapted freshwater fishes, especially their populations along warm‐range margins, are most vulnerable to the climate oscillations associated with global warming. Stocking is a major strategy for avoiding the extinction of these species. However, while stocking can reverse the decline of isolated populations, it may also result in a loss of genetic diversity in the native local population due to the introgressive replacement of hatchery genes. To plan an adequate strategy for conserving locally adapted populations, the genetic impacts of stocking on native lineages should be evaluated from small river branches to wide‐ranging drainage areas. We investigated the population genetic structure of white‐spotted charr (Salvelinus leucomaenis) within its southern range (Lake Biwa basin, Japan). By applying genome‐wide SNP analysis to the population's genetic structure, we assessed the extent of genetic introgression resulting from stocking. White‐spotted charr in the Lake Biwa watershed constitutes a distinctive genetic group, within which apparent genetic differentiation was observed. The hatchery‐reared fish line commonly used for supplementation stocking in the catchment was discernable from the native population, enabling us to analyze genetic introgression across the entire drainage area. Admixed individuals resulting from hatchery introgression were observed in most of the stocked sites that showed relatively high heterozygosity and nucleotide diversity. However, their genetic differentiation was much lower than that of native populations. The supplementation history as well as the road availability contributed substantially to the introgression of hatchery genes. Populations with the native genetic structure remained in the upstream regions of the tested rivers. However, their heterozygosity and nucleotide diversity were low when compared with that of the populations with hatchery supplementation. Our results shed light on the genetic impacts of stocking on isolated native populations and suggest that conventional supplementation methods cannot preserve a unique biodiversity in the distribution margin.