This chapter provides detailed descriptions of the protocol used for fish sperm cryopreservation. The principles described can be applied to any species, but details are also given for individual fish species. Successful cryopreservation of fish spermatozoa depends on a range of factors including the collection of high quality sperm, equilibration conditions, choice of cryoprotectant medium, cooling/thawing regimes, and conditions for fertilization. Even though some general rules can be applied to any fish species, optimization of the protocol is needed for each individual species. Furthermore, because sperm derived from marine and freshwater fish differs markedly, the described protocol is mainly focused on the cryopreservation of sperm for freshwater fish because this poses the greater technical challenge.