Indian mustard (Brassica juncea L.) is an important oil seed crop. The present investigation was undertaken to explore and detect genetic diversity of five mustard accessions i.e. Maya, Kranti, Poosa Bold, RH- 30, and RGN-73 by SDS-PAGE for protein profiling and Randomly Amplified Polymorphic DNA (RAPD) for assessment of genetic variation at the molecular level. Gel were scored for the presence (1) and absence (0) for polymorphic band. Molecular diversity studies were also done to find out the maximum polymorphism of DNA. The most efficient amplification and polymorphism of DNA was found with random primer OPA-11, 5’- CAA TCG CCG T- 3’.Total 8 polypeptide bonds were distinguished among five accession. Out of these 4 band was found polymorphic with a polymorphism of about 0.83% similarity coefficient. The average polymorphism was calculated 50%. The similarity matrix computed with Jaccard’s coefficient revealed the maximum similarity between accessions Poosa bold with RH-30 and Maya with Kranti, while distantly related varieties where observed with RH-30 and RGN-73. Brassica cultivars RH-30 and RGN-73 were found to produce hybrid vigour during breeding programs because they are genetically distinct from other cultivars of Brassica and have minimum genetic similarity