Heavy metal accumulation in agriculture land is one of the major agriculture challenges affecting the crop production and threatens the food safety. Comparative study for Lead (Pb) tolerance in three sugarcane (Saccharum officinarum L.) genotypes was carried out under in vitro conditions. Fresh weight, dry weight, callus morphology, shoot morphology, protein contents, catalase, superoxide dismutase and peroxidase, ascorbic acid contents, relative growth rate, stress tolerance index, Pb contents, bioconcentration factor were compared at 6 different concentrations of Pb (0mM, 0.1, 0.2, 0.3, 0.4, 0.5, 1mM). Lead affected the callus and its regeneration in all three genotypes while root regeneration did not affect considerably. All genotypes significantly vary at each treatment and parameter except BCF and protein contents. Genotype YT-53 proved most resistant toward Pb stress while CP-77400 was next to it and NSG-59 showed least resistance toward Pb stress. Most resistant genotypes can be used in heavy metal effected area for better yield and phytoremediation in Pb affected soil and in other breeding programs.