The bagrid catfish, Pseudobagrus ussuriensis, exhibits significant sexual dimorphism in growth rate and body size with males growing faster than females. Therefore, an all-male culture can dramatically increase the output and profitability of fishery products. According to the monosex breeding route, super-male individuals’ acquirement by XY male sex reversal and artificial gynogenesis is the key step. An effective protocol to induce meiotic gynogenesis using homologous sperms has been developed in this study. The most effective UV irradiation for sperm genetic inactivation was found to be at a distance of 20 cm with 66 μW/cm2 light intensity for 25 min. Optimal treatment for cold shock was 5 min post-fertilization at 0-4°C for 30 min, which gave the best survival rate of 13.65 ± 2.87%. The sex ratio in the meiotic gynogens showed a significant female-biased deviation (p < .05); thirty meiogynogens and their parents were further analysed using a male-specific AFLP marker, of which only the male parent produced a male-specific DNA band of 412 bp. These results indicated the female homogametic XX/XY sex determination system in P. ussuriensis. The optimization of a protocol for the successful induction of meiogynogenesis in the bagrid catfish lays the basis for all-male production and is useful in ascertaining the genetic sex determination system in this promising aquaculture species.