HPLC analysis of Echium plantagineum seed oil shows a complex triacylglycerol (TAG) profile. TAG species were separated on an analytical scale by HPLC and their fatty acid (FA) composition is reported. GLC analyses showed that some TAG fractions reached a stearidonic acid (SDA, 18:4n-3) percentage significantly higher than that in the original oil. TAG separation on a bigger scale was also essayed, by means of a gravimetric normal-phase chromatographic column, using silver ion-silica gel as stationary phase. Gradient elution with solvents of increasing polarity was applied, allowing the separation of valuable TAG species containing γ-linolenic acid (GLA, 18:3n-6), α-linolenic acid (ALA, 18:3n-3) and SDA as the main constituents (more than 85% of the total FA). An enzymatic hydrolysis reaction showed the distribution of FA in the isolated species of TAG. SDA was the major FA in the sn-2 position (more than 50% of total FA), followed by ALA (19%) and GLA (18.5%).