Due to the climate change and the genetic erosion of crops over time, the strategy of replacement of tolerant species and the use of wild species as an important and rich source of useful genes seems to be necessary in breeding programs. For this purpose, in this study, drought tolerance and genetic variation in F 5 lines from cross-species of safflower including crosses between Cartamus tinctorius × Cartamus palestinus (IP), C. palestinus × C. oxyacanthus (PO) and C. tinctorius × C. oxyacanthus (IO) were investigated in two non-stressed and drought stressed environments as a simple 14×14 lattice design. The results showed that, for most traits, the F 5 lines have been roughly purified enough allowing line selection in this germplasm. The results showed that drought stress had a significant effect on days to button, days to flowering, days to maturity, number of capsuled per plant, seed yield and oil percentages. Drought stress, had lowest effect are days to button (1.79) % reduction and the highest effect on (71.07) % reduction. There was a significant difference between the F5 lines for all traits except days to maturity. Drought stress did not reduce genetic diversity. The results of the comparison of the mean of different traits showed that, on average, IP lines in the non-drought stress had the highest yield and oil content, but in the drought stress, the highest yield and oil, as well as the lowest percentage of reduction of most traits, were observed for PO and IO crosses indicating that these crossings had more sustainability. Correlation results showed that there is a possibility of indirect selection for improvement of yield through some traits such as number of capsule per plant. Based on the principal component analysis (PCA) at the IP cross in the non-stressed condition genotypes 12, 5, 48, 6 and 8 and in the drought stress condition, genotypes 47, 48, 16, 21, 27, 46, 12 and 2, were introduced as superior genotypes. In the PO cross in the environment the non-stress condition of genotypes 74, 81, 78, 112 and 95 and in the drought stress of genotypes 93, 94, 79, 92 and 116 were introduced as superior genotypes. In the IO cross in non-stress conditions genotypes 137, 139, 136 and 142 and in drought stress condition genotypes 164, 179 and 180 were introduced as superior genotypes. Based on drought tolerance indices, PO and IO crosses showed more stability in stress conditions. IP crosses showed the most susceptibility to stress. Thus, genotypes 72 and 164 were tolerant. Genotypes 152, 93, 120, 49, 106 and 59 had yield stability. Finally genotypes 14, 48, 68 and 115 were introduced as high yield and tolerant genotypes. Keywords: F 5 generation, Drought Stress, Genetic Diversity, Safflower.