Buglossoides arvensis is indigenous to India and its seed oil is rich in unique and nutritionally important omega-3 fatty acid namely, stearidonic acid (SDA). It is a non-conventional oil seed plant and needs to be agronomically adapt for commercial utilization. In the present study, oil extracted from the agronomically adapted high yielding cultivar of B. arvensis seeds was analyzed for its oil content, fatty acid (FA) composition, physico-chemical characteristics, total lipids and triacylglycerol molecular species. The oil content, peroxide, acid, iodine, p-anisidine values and tocopherol content of the oil were 18.53% (w/w), 2.06 meq of active oxygen / kg of oil, 2.55 mg KOH/g oil, 217.2 g I₂/100g oil, 10.7 and 774.8 mg/kg oil respectively. Oxidative stability as determined by the induction period was found to be 3.1 h. Polyunsaturated fatty acid (PUFA) content of the oil was 81.3% (of total FA), comprising of α-linolenic acid (ALA; 48.5%), SDA (18.6%), linoleic acid (LA; 10.3%) and γ-linolenic acid (GLA; 3.9%). Profiling of lipid classes showed neutral lipids (89.3%, w/w) as most abundant lipid class followed by glycolipids (7.4%, w/w) and phospholipids (3.3%, w/w). High resolution mass spectrometric analysis of triacylglycerol (TAG) molecular species showed TAGs with C54 carbons in the acyl chain as most abundant. Positional distribution analysis showed GLA and SDA predominantly at the sn-2 position of triacylglycerol. FTIR analysis revealed common characteristics molecular features similar to PUFA rich oils. Overall, the results suggest that B. arvensis seed oil is an excellent ω3-ω6-ω3 or ALA-SDA-GLA source for food and nutraceutical industries.