During 2021 through 2023, the golden mussel Limnoperna fortunei and freshwater fishes were sampled from 28 sites in the Tone River system, Japan, and adult trematodes of Dollfustrema were found in the fishes. Molecular and morphological analyses based on 28S rDNA and the ITS1−5.8S−ITS2 region revealed the trematode as ‘Dollfustrema hefeiense’, previously reported in Mainland China and likely introduced to Japan. Given that its scientific name was considered invalid, we re-described the species as Dollfustrema invadens n. sp. Additionally, the DNA-based survey helped clarify the trematode’s life cycle in the river system. A sporocyst and metacercariae were detected in the golden mussel’s visceral mass and in the muscles of two small freshwater fish species, respectively. The channel catfish Ictalurus punctatus harboured mature trematodes in its intestine, and adult trematodes were also found in the muscles of fishes infected with metacercariae, suggesting direct metacercariae development in fish muscle. Furthermore, another introduced bucephalid trematode, Prosorhynchoides ozakii, previously reported in the river system, was detected in the mussels and fishes. Moreover, co-infection of both bucephalid trematodes was observed in certain fishes.