) grow faster than males. Therefore, farming all-female progenies could maximize profits from cultured fish. In the present study, diploid gynogenesis was induced by activating flounder egg development with UV-irradiated (72 mJ/cm²) sperm for 3 min in sea water, and then subjecting the eggs to a cold-shock (0–2) treatment for 45 min. Cold shock was used to prevent extrusion of the second polar body. In the control treatments, one or more of these steps was omitted to separately assess the effectiveness of UV irradiation and cold shock. When the initial treatment was applied 3 min after hatching (treatment duration, 45 min), the fertilization rate of eggs activated by UV-irradiated heterologous sperm was (37.2 ± 5.1)%, the malformation rate of newly hatched larvae was 8.3 ± 2.5)%, and the hatching rate was up to (9.4 ± 0.71)%. Flow cytometry and chromosome karyotyping were used to examine the ploidy level of larvae experienced to the cold-shock treatment. All of the gynogenetic larvae were diploid (2n = 48), and no haploid or aneuploid phenomena were detected. The duration of hatching of haploid embryos and gynogenetic embryos was significantly different from that of control normal embryos. There were no significant differences in the morphological index between gynogenetic diploids and normal larvae (>0.05). All of the haploid larvae (100%) showed abnormal external morphology (<0.05) while normal diploid larvae and gynogenetic diploid larvae showed normal morphologies. In summary, these methods resulted in successful diploid gynogenesis in southern flounder using heterologous sperm. This technique could serve as a useful tool to produce monosex progenies of southern flounder.