Abstract Amphiploids were produced from pentaploid hybrids between Vaccinium corymbosum (4x) and Vaccinium ashei (6x) by colchicine treatment. Seeds of each parental species and those obtained by the interspecific crossing were treated with colchicine at 0, 500, 1000 and 2000 mg/l for 7 days, and the ploidy level of the seedlings was determined with flow cytometry. Either amphiploid (10x) or ploidy chimera (5x + 10x) was obtained from interspecific hybrid seeds treated with all colchicine concentrations, while no chromosome-doubled plants were obtained from both parents. Cross direction in interspecific hybridization affected the results of colchicine treatment and amphiploids were obtained only when V. corymbosum was used as the seed parent. In this cross, 17–25% of the seedlings turned to be amphidiploids or ploidy chimeras by treating with 500 mg/l colchicines for 7 days. These results indicate that susceptibility to colchicine may increase in the interspecific hybrid compared with the parental species but only when V. corymbosum was used as female parent.