The quality of oil in Brassica juncea is significantly influenced by its erucic acid content, highlighting the need for developing low-erucic acid genotypes through Marker-Assisted Selection (MAS). This study aimed to characterize 175 segregating F₃ genotypes derived from Indian genetic backgrounds from cross between Indian (Pusa Mehak) and East European (Primus) parents using Cleaved Amplified Polymorphic Sequence (CAPS) markers followed by restriction digestion for Fatty Acid Synthesase (FAE) gene’s two paralogs- FAE1.1 and FAE1.2 genes, which govern erucic acid biosynthesis. The genotypic classification based on amplification and restriction digestion revealed six distinct allelic configurations, with FAE1.1 exhibiting a stronger correlation with erucic acid content than FAE1.2. To validate these genetic findings, the biochemical profiling of oil samples by gas chromatography confirmed a bimodal distribution of erucic acid (17.45–50.92 %), with homozygous recessive e₁e₁ genotypes displaying significantly lower erucic acid (22.26 % and 24.44 %) compared to heterozygous and dominant homozygous genotypes (27.29–45.20 %). However, FAE1.2 did not show a clear association with erucic acid content, indicating its limited contribution to the trait. Gene FAE1.2’s limited role in determining erucic acid levels compared to FAE1.1, could shift selection priorities towards focusing on FAE1.1, making it the primary target for MAS in breeding programs. The study validated MAS as an efficient strategy for early-stage selection of low-erucic acid genotypes, potentially reducing breeding cycles and accelerating the development of superior Indian mustard varieties. The identified seven low-erucic acid genotypes hold promise for breeding programs aimed at improving oil quality. The integration of MAS with morphological and physiological selection criteria could further enhance breeding efficiency and facilitate development of high-yielding, nutritionally improved B. juncea varieties.