Twelve genotypes of bivoltine silkworm (Bombyx mori L.) were studied for genetic divergence using six biochemical markers namely, Aspartate aminotransferase, Alkaline phosphatase, Amylase, Esterase, Succinate dehydrogenase and peroxidase. The biochemical analysis was done by native-polyacrylamide gel electrophoresis. A total of thirty three bands were observed for the six isoenzymic systems among which six were monomorphic and twenty seven were polymorphic. However, none of the isozymes differentiated all the lines but a combination of two or more isozymes could be used to characterise them. The banding pattern of all the six isozymes showed a wide range of both quantitative and qualitative polymorphism. All these isozymes showed an appreciable range of relative mobility ranging from 0.39-0.70 in alkaline phosphatase, 0.43-0.72 in amylase, 0.31-0.73 in aspartate amino-transferase, 0.68-0.94 in esterase, 0.39-0.94 in peroxidase and finally 0.41-0.78 in succinate dehydrogenase. Agglomerative hierarchical clustering of the biochemical data classified twelve genotypes selected from among the D2 clusters into two major groups. The first group included all medium to low yielding genotypes while second group contained all high yielding genotypes. These two groups were then further classified into five sub-groups. Sub-group 1 contained NB4D2 and SH6, sub-group 2 had Meigitsu, CSR4 and Sheiki II. The genotypes Pampore-5, J122, J2M and 14M constituted sub-group 3. JBEL and NJ3 were categorized in sub-group 4 and B38 was placed in sub-group 5. The maximum distance was found between genotypes NB4D2 and NJ3 while J122 and 14M were the nearest neighbours.