Triacylglycerols (TAG) in viper bugloss oil were isolated from raw pressed oil by silicic acid column chromatography. The obtained blend of TAG was separated by silver ion thin-layer chromatography (TLC Ag 1 ) into nine fractions, varying in terms of unsaturation level and molecular polarity. The composition of TAG in viper bugloss oil was determined by HPLC coupled with a diode-array detector and an evaporative light-scattering detector. The results showed that the first three fractions were combinations of TAG containing palmitic, oleic and linoleic acids. Fractions 4 and 6 contained TAG of a similar acid composition as above, but with the addition of g-linolenic acid. The remaining fractions (7-9) were the most varied in acid composition. They were found to contain 26-39% palmitic acid, 12-15% oleic acid, 13-41% linoleic acid 8-24% g-linolenic acid, 1.5-5.5% a-linolenic acid and 1-5% stearidonic acid. The analysis of fatty acid allocation in TAG of viper bugloss lipids revealed that linoleic acid (ranging from 2 to 100%) was the only acid found in all isolated fractions. In the investigated oil, the predominant TAG included: LnLnG (11.38%), LnLnSt (11.17%), LnGSt (7.71%), LnStSt (6.19%) and LnLnLn (5.44%). Almost 86% of the TAG contained a-linolenic acid, while g-linolenic and stearidonic acids amounted to 49 and 38%, respectively.