Abstract: The long period of sexual maturity and the characteristics of hermaphroditism extend the breeding cycle of grouper. In addition, the artificial gynogenesis strategy can significantly improve the breeding efficiency. In this study, we explored the best conditions for artificially inducing gynogenesis in Epinephelus coioides used by the sperm of Epinephelus lanceolatus, and were identified by morphology, flow cytometry and microsatellite markers. The results showed that artificial gynogenesis of Epinephelus coioides was successfully induced by inactivated heterologous sperm. The best suitable UV irradiation intensity of sperm was 518 mJ/cm², and the irradiation time was 4min. 6min after fertilization, cold shock treatmentcould obtain the optimal apparent fertilization rate of 40% and hatchability of 29.7% at 4—6℃, while the control group without cold shock showed 83.5% apparent fertilization rate and 80% haploid rate. The result of multiple comparison showed that in terms of apparent fertilization rate, there is no significant difference between the artificial induction time (P=0.579) and water temperature (P=0.133). When it comes to hatch rate, the difference of treatment time between groups is extremely significant (P=0.002), but not in water temperature group (P=0.396). The results of embryo development showed that the induction of artificial gynogenesis would lead to the decrease of partial hatchability and the haploid embryos behaved obvious developmental disorder. The results of microsatellite molecular markers indicated that most of the genetic material of the artificial gynogenetic progeny of grouper came from the female parent, and the heterospermia effect also exists.