Mutation using radiation has been shown to be an important tool in incorporating specific desirable agronomic value characteristics. In the present study, four grapevine rootstocks namely: Freedom, Harmony, SO4 and Ramsey along with commercial variety Thompson seedless were exposed to different doses of gamma rays (0, 10, 20 Gy) to obtain mutations with economic values.Results showed that both doses of 10 and 20 Gy were effective in obtaining mutations in all grapevine genotypes including Thompson seedless, which were confirmed by morphological markers such as bud burst percentage and shoot length and number of leaves, and genetic markers by RAPD technique.In addition, genetic variation and relationships in both irradiated and un-irradiated genotypes were determined. Six RAPD markers were able to generate polymorphic bands ranged from 1 to 9 among irradiated and un-irradiated grapevine genotypes. The similarity coefficients detected by RAPD markers ranged from 0.24 to 0.87, which revealed clear genetic variation among irradiated and un-irradiated grapevine genotypes. According to phylogenetic analysis, genotypes were divided into two clusters, where all five un-irradiated genotypes and irradiated Freedom genotype with 10 Gy were placed in one cluster and second cluster included the rest of nine irradiated genotypes.Conclusively, this study supports the use of mutation in breeding grapevine improvement, and RAPD technique could be an important tool for detecting mutations and genetic diversity among grapevine genotypes.