Milk polar lipids are an important class of biologically active species for human health and for improving the physical functionality of food ingredients. Milk polar lipids from 144 multiparous Holstein-Friesian dairy cows fed different diets were analysed using ultra-high-performance liquid chromatography-ion trap mass spectrometry (UHPLC-MS(n)). A complex profile of polar lipids, consisting of 7 species of phosphatidylinositol (PI), 12 species of phosphatidylethanolamine (PE), 18 species of phosphatidylcholine (PC) and 13 species of sphingomyelin (SM) were identified from the molecular ions and sequential MS(n) fragmentation. Qualitative assessment of the data suggested that different cow diets influenced the relative amounts of a small number of species in the milk samples, e.g. PE 14:0/18:1, PE 18:0/18:1, PC 15:0/18:1, PC 18:0/18:1, SM d18:1/14:0, SM d18:1/15:0, SM d18:1/22:0 and SM d18:1/23:0.