Abstract: Sugarcane is an important crop in Indonesia. Protocols were established to induce callus and treat with Ethyl Methane Sulphonate (EMS). Calli were induced and immersed in 0.1, 0.3 and 0.5% EMS solutions for 30, 60 and 120 min. PS 862 and VMC 7616 sugarcane varieties were used in this experiment. Mutant putative calli were selected on media with aluminium concentrations of 100, 200, 300, 400 and 500 ppm and then cultured on regeneration media. Sugarcane mutant plants were evaluated for its aluminum tolerance by planting them in acidic soil (pH of 4.1). This research showed that increasing EMS concentration reduced the regenerative capacity of sugarcane calli. Calli of PS 862 had a higher aluminium tolerance capacity compared to calli of VMC 7616. Mutant putative shoots could be induced to produce roots by treatment with 5 mg L^–1 IBA. Field evaluation showed that mutant clones had higher agronomical characters value than mother plants which was due to the EMS-induced genetic mutation. Mutant clones had plant heights in the range of 2.8-4.3 m, the number of shoots 3-12, the number of nodes 9-16 and stem diameters 1.3-2.8 cm. Meanwhile, PS 862 and VMC 7616 (mother plants) had plant heights 2.9 m and 2.97 m, number of shoots 3.7 and 1.67; nodes number 8.14 and 7.33 and stem diameters 1.27 and 1.33 cm. The Brix values of mutant clones were in the range of 12-20% and fresh weights were 900-2610 gr while brix content of PS 862 and VMC 7616 (mother plants) were 16 and 15% and the fresh weight were 1419 and 1650 g.