Japanese flounder (Paralichthys olivaceus) is a widely cultured marine fish species in China and is high valued because of its fast growth and good taste. However, diseases of the cultured fish have occurred frequently and losses due to infectious diseases limit profitability and development of aquaculture. To breed new flounder strain with enhanced disease-resistance and growth rate is an important task in marine fish aquaculture. In the present study, 94 disease-resistant individuals selected from natural selection and artificial challenge by pathogenic bacteria, Vibrio anguillarum, were used as one of basic populations for developing family. Japanese population of the flounder were imported from Japan in 2003 and used as one of basic populations. In addition, the flounder captured from Yellow sea were used as one of the basic population for developing family. Different mating of the above three populations resulted in 63 families in which 54 families are half-sibling family, 9 families are full-sibling families. Growth comparison demonstrated that there are big differences in growth rate among the 63 families. Some families exhibited fast growth while some families grow very slowly. Among the 63 families, 4 fast-growth families (Family 36, 42，43 and 57), 9 secondly fast-growth families（Family 27、28、35、39、41、51、60、65 and 76）, and one slow-growth family (75) were identified. Bacterial challenge experiments demonstrated that there are big differences in disease resistance among the 59 tested families. Some families exhibited strong disease resistance to bacterial infection, while some families are not resistant to bacterial challenge. Among the 59 families, 3 families (Family 50,61 and 68) with strong disease resistance with a survival rate of over 60% were identified, 17 families with secondly strong disease resistance with a survival rate of 39%-60%, 33 families with normal disease resistance with a survival rate of 17%-39%, and 6 families with low disease resistance with a survival rate of less than 17% were identified. Development of the 63 families lay foundation for selective breeding of flounder stains with fast growth and disease resistance.