Abstract: Mass propagation of sugarcane through meristem culture ensures quick availability of the genetically uniform virus free newly released varieties to the farmers but the occurrence of genetic variation however, limits its commercial application. Isoenzyme analysis may be used to screen somaclones to maintain varietal purity of a sugarcane hybrid like that of a breeder seed in conventionally propagated variety which will be of great benefit to the farmers. Organogenic plant regeneration directly as well as via callus induction from shoot tip of sugarcane indicated that the hormone treatment of KIN (2.0 mg L-1) and NAA (5.0 mg L-1) along with 3% table sugar was best for meristem tip enlargement and shoot initiation. For shoot multiplication a treatment combination of BAP and KIN (1.5 mg L-1 each) was found to be optimum. Highest number of shoots was obtained in treatment combinations of BAP and KIN (1.5 mg L-1 each) along with GA3 (3.0 mg L-1). NAA (5.0 mg L-1) was found optimum with high mean response for rhizogenesis. The detection of genetic variability by analysis of peroxidase isozyme of mericlones and calliclones indicated presence of polymorphism within the mericlones and calliclones. The mericlones exhibited genetic integrity for invertase isozyme while exhibiting the lowest polymorphism for total soluble proteins. The calliclones regenerated after third callus subculture exhibited lowest polymorphism over peroxidase, invertase and total soluble proteins. Thus, micropropagation by callus culture can be advanced up to third subculture to obtain large number of plantlets with minimum variability from that of the parental genotype.