Sperm cryopreservation is a widely employed technique that promotes alternative techniques to contribute to broodstock management or restoration programs for species of commercial interest, endangered species or species with an interesting genotype. The preservation of genetic material from improved stocks or from the original population is extremely important for the oyster aquaculture industry to prevent the potential impacts of epidemic diseases and natural disasters. The Portuguese oyster, Crassostrea angulata, was the most important species commercialized by the shellfish industry. However, inadequate management of this industry and pathology occurrences resulted in a significant decrease in natural populations. For this reason, in this work a successful sperm cryopreservation protocol for this important species has been developed for the first time. Different internal cryoprotectants (DMSO, ethylene glycol, polyethylene glycol and methanol) at several concentrations (5, 10, 20%), containers (straws vs cryovials) and freezing rates (slow and fast rates) were tested. Cryoprotectant toxicity tests corroborated that this assay did not take into account the following steps of cryopreservation protocol as sperm agglutination. A fast freezing rate of cells diluted in10% DMSO and the use of straws as containers were the best cryopreservation conditions for Portuguese oyster sperm. Finally, fertilization assays confirmed the efficiency of the cryopreservation protocol in oyster sperm. These results demonstrated that different susceptibilities have been detected concerning sperm cryopreservation depending on oyster species or genetic material composition.