The genetic improvement of rice through mutation induction to obtain drought resistance is essential for stable and adequate rice production would enable us to anticipate the rice yield-reducing effects of global warming. The aim of this research was to evaluate the rice mutant lines showing tolerance to drought condition and their genetic relationship using SSR markers. Mutant lines were obtained by irradiation of Mira-1 rice variety at primordial state by 25 and 50 Gy doses. Twenty numbers of M4 rice mutant lines, cv. Mira-1 as wild type, cv. Gajah mungkur as positive control and cv. IR64 as negative control were chosen in this study were planted in PVC cylinders in the green house. The result showed that the agronomic trait of rice mutant lines were better than their wild type. Root length of 17 number of mutant lines are longer than their wild type and Gajah mungkur, the longest root obtain was 71.5 cm on 7J4 rice mutant line compared to Mira-1 only which was 25 cm. The number of lateral roots and seeds content per panicle higher than their wild type and positive control. Based on SSR marker linked to root depth, DRO1 showed that 12 numbers of mutant lines polymorphic and changing of genetic of mutant lines which was indicating the mutant lines were tolerant to drought compared to their wild type. The molecular information of rice mutant lines is essential for further development of a new superior variety of rice, which would be an improvement of the variety already existed.