Indian mustard (Brassica juncea L.) is an important source of edible oil in India. Quality parameters such as moisture, oil, protein, erucic acid and sinigrin were analysed in sixty genotypes of mustard (Brassica juncea L.) and from which 15 genotypes with desired proximate parameters were selected and analysed further for anti nutritional factors, genetic diversity and isozymes. Erucic acid and Glucosinolate (Sinigrin) was analysed by gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography (UPLC), respectively. SKM 9033 showed the highest (30.88%) oil where as RAYAD-9602 had the lowest (20.80%) oil. The protein content and true protein in mustard cultivars ranged from 27.90–33.69% and 10.66–20.01%, respectively. HNT-33 genotype had the highest concentration of erucic acid where as NRCM120 showed the lowest. Bio-Q-44-279 had the highest sinigrin concentration (46.80 μmole/g) while SKM-9033 had the lowest sinigrin concentration (2.49 μmole/g). Isozyme pattern of peroxidase and esterase showed significant correlation amongst the clusters. Electrophoresis of seed protein by SDS-PAGE showed a total of 28 bands with varied intensity in mustard genotypes. Both Simple Sequence Repeat (SSR) and Inter Simple Sequence Repeat (ISSR) markers were found to be efficient to assess genetic diversity within species of mustard with average polymorphism of 82.91% and 96.02%, respectively. NRCM-120 and SKM-9033 had low content of erucic acid and sinigrin respectively and hence were found to be useful for the development of double zero variety by breeding program. The SW-1-9017 and Bio-341-92 genotypes showed high genetic diversity compared to other genotypes and found to be useful for breeding purpose.