Tetraploid fish are important for mass production of triploids in polyploid breeding.In this study,we reported a novel protocol for artificial induction of tetraploidy by a combination of cold shock and hydrostatic pressure administered to gynogenetically developed eggs in Japanese flounder Paralichthys olivaceus.The induction was carried out by activating the eggs with UV-irradiated sperm of red sea bream Pagrus major,administering a cold shock (0℃,5 to 45 min) 3 min after fertilization to inhibit second polar body exclusion,incubating the eggs for 60 min at 17℃,and treating them with a 650 kg/cm2 hydrostatics pressure shock for 6 min.We named the embryos gyno-tetraploids that developed from eggs after such treatments.The hatching rate of the gyno-tetraploids ranged from 20.99％±3.66％ to 36.01％±2.79％,and the tetraploid rate ranged from 80.00％ to 100.00％.All-matal inheritance was verified using 6 high-recombination-rate microsatellite markers.This method successfully induced gyno-tetraploidy.The successful induction of gyno-tetraploidy lays the foundation for triploidization of new varieties with improved economic traits of interest that can benefit commercial culture.