Hybridization is one of the negative outcomes for the introduction of non-native species, which can lead to rapid displacement and genetic extinction of native species. Salmonid fishes have been widely introduced outside of their native ranges for food supply and recreational fishing. Here, we investigate the occurrence of introgressive hybridization among native Dolly Varden (Salvelinus curilus (syn. Salvelinus malma)), white-spotted charr (Salvelinus leucomaenis), and introduced brook trout (Salvelinus fontinalis), in streams of the Nishibetsu River, Hokkaido, Japan. Microsatellite DNA analysis detected five hybrids between native Dolly Varden and introduced brook trout. This is the first evidence for hybridization between native Dolly Varden and introduced brook trout, while the latter has been known to hybridize with many other salmonids. Furthermore, incongruence between mitochondrial DNA and microsatellite DNA analyses suggested introgression among the three Salvelinus species. Further studies to estimate the hybrid fitness are necessary to understand how hybridization among the three species affects the native species.