The combinations of different concentration 6-BA and NAA were used to screen appropriate culture medium for adventitious buds induction of Amomum villosum Lour. The induced buds were radiated by 60Co-γ ray with different irradiation doses and ISSR molecular marker was used to analyze the genetic diversity of the mutants. The optimum culture medium for adventitious buds induction was MS+6-BA 4.0 mg/L+NAA 0.1 mg/L, in which the maximum buds induction rate of 91.4% and multiplication coefficient of 2.44 were attained. The appropriate dose of γ ray for buds was 16 Gy. Two plants irradiated with 16 Gy dose, AV16-1 and AV16-2, were mutated significantly according to the genetic similarity coefficients, which were 0.549 and 0.563, respectively. The breeding method of A. villosum by combining tissue culture and radiation mutagenesis to generate new germ plasm was established by this research.