Abstract: To study the genetic variation degree of plant characters of Dendrobium tissue culture seedlings treated by radiation mutation, in this study, SRAP molecular markers technique was used to analyze Dendrobium sonia‘166’variety tissue culture seedlings treated by different doses of 60Coγ ray. The radiation mutagenesis effect was discussed, and the optimal irradiation dosage was determined. The results showed that 12 primers screened out from 100 pairs primers were used to amplify the genomic DNA of Dendrobium treated with different doses of 60Coγ ray. The 216 bands were amplified, among them 41 bands were polymorphic with the ratio of 19.0%. The cluster analysis of the bands amplified in treated and controlled plants showed that D. sonia‘166’varieties tissue culture seedlings treated by different doses of 60Coγ ray mutated in different extent. The variation degree was maximum in 20 Gy treated plants, and was minimum in 5 Gy treated plants. Therefore 20 Gy was the best radiation dose of the 60Coγ-ray for variation induction.