Abstract: Tetraploid of Crassostrea gigas "Haida No.2" was induced by cytochalasin B (CB) and hypotonic treatment under pathway Ⅰ (2n♀+2n♂), pathway Ⅱ (3n♀+2n♂) and pathway Ⅲ (2n♀+4n♂), and this study compared the three pathways in the aspects of cleavage rate, D larvae rate, tetraploid rate of larvae and spat. The effects of three factors including reagent treatment intensity, induced occasion and induced duration were also discussed. The results showed that under pathway Ⅰ (2n♀+2n♂), the highest tetraploid rate of 28.37% was obtained in larval stage treated with 0.5 mg/L CB, and 20 min for duration time of induction when the first polar body of fertilized eggs appeared. Under this pathway, tetraploid larvae could not be detected using hypotonic treatment. Under pathway Ⅱ (3n♀+2n♂), the highest larval tetraploid rate of 52.64% was obtained in larval stage treated with 0.5 mg/L CB, and 20 min for duration time of induction when the first polar body of fertilized eggs appeared. Under this pathway. Under this pathway, when the first polar body of fertilized eggs appeared, the highest larval tetraploid rate of 26.64% was obtained after 20 min of treatment using seawater with a salinity of 8. Under pathway Ⅲ (2n♀+4n♂), the highest larval tetraploid rate of 60.45% was obtained in larval stage after 20 min of duration time with 0.75 mg/L CB when 30% PB1 appeared. Under this pathway, the highest larval tetraploid rate of 25.51% was obtained after 20 min of treatment using seawater with a salinity of 8 when 30% PB1 appeared. Under the optimal induction conditions, the ploidy of the spats (60 days) was analyzed by flow cytometry to determine their composite ploidy under optimal induction conditions. The tetraploid rate of the spats in the CB-treated group under pathway Ⅰ (2n♀+2n♂) was 4%, which was a decrease of 24.37% compared with that of the larval stage (28.37%); no tetraploid juvenile shells were detected in the hypotonic treatment group. The tetraploid rate of the spats in the CB-treated group and the hypotonic treatment group reached 80% and 30% under pathway Ⅱ (3n♀+2n♂), which decreased by 27.36% and 3.36%, respectively, compared to the larval stage (52.64% and 26.64%, respectively).The tetraploid rate of the spats of CB-treated and hypotonic treatment groups under pathway Ⅲ (2n♀+4n♂) reached 10% and 4%, which was a decrease of 50.45% and 21.51%, respectively, compared with that of the larval stage (60.45% and 25.51%, respectively). Comparing the three pathways and two induction methods, the best choice for the production of tetraploid of C. gigas "Haida No.2" is to use CB to induce tetraploid through pathway Ⅱ (3n♀+2n♂).