Ameliorating role of ascorbic acid against Pb toxicity in three genotypes of sugarcane (YT-53, CP-77-400, NSG-59)under six different concentrations of Pb(NO3)2 (control, 0.1, 0.2, 0.3, 0.4, 0.5, 1mM) was studied under in vitro conditions. The morphological parameters like callus fresh weight and dry weight, shoot and root length, number of shoots and roots per cultured plant and biochemical parameters included antioxidants enzymes, total protein contents, ascorbic acid, Pb contents in Pb treated calli were compared with 0.5mM ascorbic acid pretreated calli at each level. It was observed that all the morphological parameters were negatively affected by Pb stress but ascorbic acid pretreatment recovered this damage. Biochemical parameters like antioxidants enzymes activity ( POD, SOD, CAT) increased under elevated Pb concentration while ascorbic acid pretreatment further enhance the POD and SOD activity while CAT activity and total soluble protein contents did not change significantly. Different genotypic behaviors towards different treatments were also observed.