Biochemical variability between four populations of Aconitum balfourii (Ranunculaceae) was studied by seed protein and isozymes electrophoretic patterns. Electrophoregram of SDS-PAGE showed a total of 20 bands with molecular weight ranging from 12 kDa to 82 kDa with difference in number of bands, band width and intensity between different populations. In the isozyme analysis four enzyme systems viz. Esterase, Malate dehydrogenase, Superoxide dismutase and Catalase were studied. 7 out of 8 resolved loci (87.5%) were polymorphic. Based on presence and absence of bands Nei and Lei’s similarity index was calculated. Similarity ranged from 64.1% to 74.4%. Matrix derived from both SDS-PAGE and Native-PAGE data were used to construct UPGMA dendrogram. The dendrogram showed two main clusters.