) is a widely cultured marine fish species in China and is highly valued because of its fast growth and good flavor. However, frequent occurrence of disease and subsequent losses limit profitability and aquaculture developmentBreeding a new flounder strain with enhanced disease resistance is an important task in marine fish aquaculture. In the present study, new Japanese flounder families were established based on Japanese stock (JS), Korean stock (KS), and stock with high resistance to (RS) by nested hybrid, cross, and gynogenesis. Forty-three and sixty-five Japanese flounder families were established in 2009 and 2012, respectively. Disease resistance was investigated using 33 families in 2009 and 43 in 2012 through intraperitoneal injection challenged with . The results demonstrate that different families exhibited variable survival rates and 13 families with high disease resistance were obtained. All 13 families exhibited significantly higher survival rates compared with the control, however, some were more significant than others (<0.05 in 10 families). Of these 13 families, three were derived from the F2 family, one from the F3 family, and two belonged to the first and second gynogenesis generations(G1 and G2). In these six families, disease resistance was high in the parents, with the exception of the F2 family; their survival rate variation coefficients were <10%. Analysis of disease resistance in the three successive generations revealed that, 13 of the 16 disease-resistance families which included 13 families mentioned above and 3 resistant families sereened in 2007 were derived from RS. In conclusion, some F2, F3, G1, and G2 families exhibited enhanced disease resistance inherited from their parents, indicating that the establishment of a disease-resistant strain based on these families is possible.