<jats:title>Abstract</jats:title><jats:p>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 thi<jats:italic>n‐</jats:italic>layer chromatography (TLC Ag<jats:sup>+</jats:sup>) 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 γ‐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% γ‐linolenic acid, 1.5–5.5% α‐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 α‐linolenic acid, while γ‐linolenic and stearidonic acids amounted to 49 and 38%, respectively.