In order to induce mutation in two commercial varieties of sugarcane of Iran, CP48-103 and CP57-614, ethyl methanesulfonate (EMS) was utilized in different doses on calli. Six callus induction treatments in Murashig and Skoog (MS) based medium were studied in a completely randomized design (CRD) with four replicates. MS based medium supplemented with 2, 4-dichlorophenoxyacetic acid (2, 4-D) in 3 mg l-1 along with 6 benzylaminopurine (BAP) in 0.1 mg l-1 was the best treatment for callus induction for both varieties. 2, 4-D in 3 mg l-1 along with BAP in 0.3 mg l-1 was also practical for callus induction of variety CP57-614. Callus cells of mentioned varieties were exposed by EMS with different concentrations. Probit analysis suggested 32.25 mM EMS for LD50. Besides it implied the significance of regression coefficient at α= 0.01. Obtained regression equation for the relation between utilized dose of EMS and mortality of calli was Y=-8.18+ 5.28 X. Five regeneration treatments in MS based medium were studied in a CRD with four replicates. MS based medium supplemented with 2 mg l-1 BAP and 0.1 mg l-1 naphthalene acetic acid (NAA) had the best results for both varieties. Five root treatments in half-strength MS medium were studied in a CRD with four replicates. Half-strength MS medium supplemented with NAA in 0.01 mg l-1 was efficient significantly for root formation of both varieties. Besides, indole-3-butyric acid (IBA) in 0.01 mg/l was also efficient for root formation of variety CP57-614. Results of simple sequence repeat (SSR) marker to prove mutation showed different patterns of polymorphism among different mutants indicating that mutation could be an efficient way to create variability particularly in vegetatively propagated plants.