Edwardsiella tarda is a significant pathogenic bacterium that causes considerable economic losses in flounderaquaculture. We created 47 flounder families using parental fish resistant to E. tarda and screened them long termthrough cross-fertilization, self-crossing, and gynogenesis experiments during April–June 2014. We infected 39 flounderfamilies with E. tarda from June–October. The half lethal bacterial concentration was determined to be 80 in eachfamily using a small number of flounder in a preliminary experiment. Survival rate was 1.19%–51.19% (mean, 20.29%).Seven families with survival rates >30% were most able to resist E. tarda; the nine families with survival rates of20.29%–30% had normal resistance to E. tarda; and the 23 families with survival rates < 20.29% were least resistant toE. tarda. Family 1406 had the best resistance, which was self-crossed with 1005. The families with the best resistancewere all descendants of families 1005, 09104, and 0915. Family 09104 was a descendant of family 0768, which wasresistant to Vibrio anguillarum, and was self-crossed with family 1005 and a Korean group. Family 0915 wascross-fertilized with Korean and Japanese groups. These disease-resistant flounder could be popularized as new strainsto reduce the occurrence of virulent ascitesosis disease.