Seeds of Lithospermum officinale L. from different climatic zones were analyzed looking for new sources γ-linolenic acid (GLA, 18:3n-6) and stearidonic acid (SDA, 18:4n-3). Cultured B. officinalis was also analyzed with comparative purposes. Analyses were conducted for fatty acid (FA) profiles in the whole seeds and in the neutral and polar lipids by GC; lipid classes by open column chromatography and preparative TLC; and tocopherols, sterols and phenolic compounds by HPLC-DAD, and the later compounds were confirmed by LC-MS. The richest GLA sample was L. officinale from St. Petersburg Botanical Garden (17.9% of total FA), while wild-growing L. officinale from the Rostov region showed the highest percentage of SDA (17.2% of total FA). Total FA content ranged from 11.3 to 20.8% of seed weight. Neutral and polar lipids accounted for ~98 and 2.27% of total lipids. Five neutral lipid classes were identified (% of NL): triterpene esters, 1.3; triacylglycerols, 93.1; free FA, 1.8; diacylglycerols, 1.4; and monoacylglycerols, 2.4. Tocopherols and sterols reached 35.7 and 83.8 mg/100 g seeds; γ-tocopherol was the main tocopherol detected, and Δ5-avenasterol was the predominant sterol. L. officinale seeds contain high amounts of phenolic compounds (389.9 mg/100 g as upper limit), in which rosmarinic acid was the main component. Overall, all data suggest the possibility of using L. officinale seed oil in pharmaceutical and cosmetic formulae and as functional food.