Tiger puffer Takifugu rubripes is one of most important fish in aquaculture. Recently, there has been progress in the production of tiger puffer aquaculture strains carrying economically desirable genetic traits. Cryopreservation techniques for the long-term preservation of their germ cells may reduce the risk of losing valuable genetic resources and the costs associated with maintenance of broodstock; however, cryopreservation methods for eggs and embryos, which contain both nuclear DNA and maternally inherited cytoplasmic compartments, have not yet been developed. In this study, we developed a methodology to produce functional gametes derived from cryopreserved testicular germ cells. Testes collected from tiger puffer were cryopreserved by a slow-freezing method, and the cryomedium for tiger puffer testis was optimized by testing several different cryoprotectants. Testicular germ cells prepared from frozen-thawed testes cryopreserved for at least two months were intraperitoneally transplanted into diploid or triploid hatchlings of grass puffer T. alboplumbeus. Transplanted germ cells migrated toward and were incorporated into recipient gonads. At 10 months post transplantation, 36.4% and 64.2% of diploid and triploid recipients, respectively, produced donor-derived sperm. In female recipients aged two years, production of donor-derived eggs was confirmed in one of 62 (1.6%) triploid recipients but not in diploid recipients. Insemination with the resultant sperm and eggs from triploid recipients generated viable offspring that originated from transplanted tiger puffer germ cells. Survival and growth potential of the offspring generated by the cross of both male and female triploid recipients were also comparable to those of control tiger puffer. This method is thus a breakthrough tool for the preservation of valuable genetic resources of tiger puffer and would help to efficiently manage broodstock with desirable genetic traits. •Current methods for preservation of tiger puffer genetic resources to improving aquaculture are inefficient.•Cryopreservation were optimized for testicular germ cells, which developed into functional gametes.•Mating of grass puffer recipients resulted in viable tiger puffer offspring that could harbor desirable genetic traits.