Mutation breeding has been generally used for the improvement of plant characters in several crops. It is a powerful and effective tool in the hands of plant breeders. In mutation breeding program, selection of an effective and efficient mutagen is very essential to produce high frequency of desirable traits. The three sugarcane clones viz; NIA-0819, NIA-98 and BL4 were selected and subjected with Ethyl methanesulfonate (EMS) doses of 5, 10, and 15 mM and untreated of each variety were used as control. The results indicated for agronomic traits showed that maximum plant height was observed (331.67 cm) in BL4 under control and minimum plant height was observed (209 cm) at 15mM in BL4. The maximum number of tillers plant-1 (7.00) at 10 Mm. The higher number of internodes was noted (16.66) at 10 mM in BL4, maximum number of internodes length (17.31 cm) at 10 mM in NIA-98 and minimum (11.91 cm) at 5 mM in BL4. The results of quality traits indicated that maximum brix (20.33 %) was obtained at 15mM in BL4 and lowest (14.66 %) in 5mM in NIA-0819. The more purity was observed (72.43) at 10 mM in BL4, fibre (15.33) at 5 mM in BL4. The maximum sucrose (14.16 %) was recorded under at 15 mM in BL4, sugar yield (6.14 t ha-1) under control in BL4 and cane yield BL4 was noted (331 t ha-1) at 10 mM in BL4. The selected sugarcane mutants were analyzed by using simple sequence markers (SSR). Out of 13 SSR primers studied, 7 primers were found to be polymorphic, whereas 6 primers were counted to be monomorphic. A total of 495 conformers were analyzed by using 13 SSR primers as presented and 495 bands, 191 bands were polymorphic, showing 38.58 % polymorphism. The maximum number of locus (20) was produced by the primer SMC-703BS and minimum number of bands (8) was produced with primer EST-SSR29. The amplification products in 10 mM (EMS) produced multiple loci, in which the total numbers of 129 scroable bands, out of which 100 were monomorphic. They showed SMC 703BS bands15 was produced with lowest number of bands (8) was obtained with primer EST -SSR 29. The primer no amplification products found in EST-SSR30-5, EST-SSR38. The amplification products in 15 mM (EMS) produced multiple fragments in which the total number of 30 scroable bands was polymorphic. Some specific bands were also identified thus reflecting the SSR application for the identification of sugarcane mutants. The results showed that 15 mM (EMS) indicated 6 primers produced a single polymorphic band and 7 primers produced two polymorphic bands. The genetic similarity indices calculated by Jaccard’s similarity coefficient varied from (0.98 to 0.85 % indicate a high level of genetic similarity among the mutants that was mainly attributed to intra specific diversity. Hence, this SSR technique helped to identify the genetic variation in mutant plants.