The assessment of genetic divergence plays a significant role to identify promising genotypes to initiate crossing programme for crop improvement. The sesame being a nutritious oil seed crop containing various biochemical constituents used as a good dietary choice. The biochemical analysis was done taking freshly harvested seeds of eight (8) parental genotypes and twenty one (21) advance lines. The studies revealed that all the genotypes differed significantly among themselves for all the biochemical parameters including moisture content, oil content, its iodine number and saponification value, both crude and soluble protein, some mineral matters - Ca, P, Mn, Zn, Cu \& Fe and Carbohydrate and ascorbic acid content. Mahalanobis generalized genetic distance using D-2 statistics for the assessment of genetic divergence amongst 29 genotypes based on biochemical parameters. All the genotypes were grouped into eleven distinct clusters. Maximum intra cluster divergence was noted against cluster IX with advance lines having black coloured seeds as its constituents. With regard to inter-cluster distance values the cluster X was mostly divergent from all other clusters and cluster II as the less divergent one from the rest. Cluster X with parental genotype B-14 was identified as superior cluster for the biochemical parameters on the basis of securing higher position for maximum number of characters. Constitution pattern clustering was independent of eco-geographical isolation. Promising segregants with higher oil yielding potentially may yield from inter se mating of advance line 14 and HT-1 with T-12, R-9 or advance lines 10,14,20.