<p>Many freshwater fish face extinctions due to habitat destruction and overfishing. Bitterlings, which lay their eggs in the gill chambers of freshwater mussels, are in steep decline worldwide, along with mussels, owing to the concreting and siltation of riverbeds. In particular, Tokyo bitterling is listed as “endangered” on the Red List and is on the verge of extinction. Although in situ and ex situ conservation efforts are underway, their effectiveness is limited. Here, we developed a method for long-term preservation of genetic resources by combining germ cell cryopreservation and transplantation. We transplanted Tokyo bitterling germ cells stored in liquid nitrogen into oily bitterling recipients lacking endogenous germ cells due to <i>dnd</i> knockdown, and the recipients matured and repeatedly produced many eggs and sperm. The resulting offspring had the Tokyo bitterling genotype and morphology. This study shows that even extinct species can be revived if their germ cell bank is available.</p>