Cryopreservation of fish sperm is a valuable method for restoration of endangered species as well as serving as complementary technique of reproductive manipulation for genetic improvement. In the cryopreservation of sperm, exposure to cryoprotectant prior to freezing is an important factor for many species. Therefore, the present study was conducted to develop an optimal freezing rate in the cryopreservation protocol of red tilapia fish sperm. The results obtained from the present study showed that the main factors that influenced the quality of sperm used in cryopreservation were molarity of glycerol in extender, equilibration time, vapour temperature and individual fish. However, in an attempt to develop an efficient and routine cryopreservation technique of fish sperm, more research needs to be conducted.