Long-term preservation of fish fertility is essential for the conservation of endangered fishes. However, cryopreservation techniques for fish oocytes and embryos have not yet been developed. In the present study, functional eggs and sperm were derived from whole rainbow trout that had been frozen in a freezer and stored without the aid of exogenous cryoprotectants. Type A spermatogonia retrieved from frozen-thawed whole trout remained viable after freezing duration up to 1,113 days. Long-term-frozen trout spermatogonia that were intraperitoneally transplanted into triploid salmon hatchlings migrated toward the recipient gonads, where they were incorporated, and proliferated rapidly. Although all triploid recipients that did not undergo transplantation were functionally sterile, 2 of 12 female recipients and 4 of 13 male recipients reached sexual maturity. Eggs and sperm obtained from the salmon recipients were capable of producing donor-derived trout offspring. This methodology is thus a convenient emergency tool for the preservation of endangered fishes.