Indian mustard is the second most important oil seed crops of India, next to groundnut sharing 27.8% in the India’s oilseed economy. The improved mustard seeds contain 39-44% oil. Oil quality is determined by fatty acid profile, whereas, level of erucic acid predicts the quality of seed oil.  Breeding of oil seed has evoked a strong bottleneck selection towards double-low (00) seed quality with zero euroic acid and low seed glucosinolate content. DNA based molecular markers are important tools in breeding programmes for crop improvement. The main role of these makers to detected the polymorphism. The experimental material comprised 71 genotypes including parents, half diallel crosses and simplified triple test crosses for study. In molecular characterization of erucic acid and glucosinolate using 4 SSR markers was clustered into three groups. Molecular analysis was done using four markers and a total of 56 amplified bands were obtained, out of which 17 were polymorphic. All the genotypes were clustered in three groups by using DARwin software. Cluster I had seven genotypes viz., RSPR-01, PM-21, PM-22, RB-50, Urvashi, Nov. Gold and Pusa-Bold followed by cluster II (five genotypes viz., RSPR-03, Varuna, Pusa-Karishma, RL-1359 and Kranti  and cluster III (two genotypes viz., PM-24 and NRCDR-2).