The total lipid fraction of eggs from hens fed diets enriched in seal blubber oil (1.25–5.0% SBO) was directly analysed with normal-phase high performance liquid chromatography coupled on-line with electrospray ionization ion-trap tandem mass spectrometry (HPLC-ESI-MS–MS) for the identification of the molecular species of phospholipids (PLs). The species of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) were all detected as the M − H − ions. The phosphatidylcholine (PC), sphingomyelin (Sph) and lysophosphatidylcholine (LPC) classes, were detected as formate adducts M + HCOO −. Tandem MS of PE and PI showed the loss of the carboxylate anions, and, for PI, also the loss of water and inositol. Product ion spectrum of PC, LPC and Sph contained only the M − CH 3 − ion fragment. Feeding different levels of SBO for 5 weeks resulted in a significant increase of PE, PC and PI molecular species carrying eicosapentaenoic acid (C 20:5 ω3 , EPA), docosapentaenoic acid (C 22:5 ω3 , DPA) and docosahexaenoic acid (C 22:6 ω3 , DHA), but not Sph nor LPC. The highest increase of the ω3/ω6 ratio occurred for PE and PC. On the contrary, PI was less affected by the increase of SBO in the diet.