Bovine milk contains 3-5% of fat, of which the dominant portion (about 98%) is in the form of triacylglycerols, whereas polar lipids such as glycerophospholipids and sphingolipids are minor components (0.5-1%) of milk fat. Milk fat is thought to contain several thousand lipid species, making it the most complex material in nature in terms of lipid composition. Significant progress has been made in the past two decades in the identification and quantification of lipid species of milk thanks to the advance in analytical tools especially high-resolution mass spectrometers (MS). Currently, milk lipids are characterized mainly in two ways, i.e. global fatty acid composition profiling by gas chromatography and lipid molecular species identification and quantification by liquid chromatography tandem MS; the former provides information related to the physicochemical properties and nutritive quality of milk fat, whereas the latter provides the detailed chemical structure of lipid species. Until now, only about 400 lipid species have been described in bovine milk, with many low-abundance species remaining to be discovered. The merits and limitations of various separation techniques and different MS methodologies applied to lipid structural elucidation and quantification are critically reviewed and the challenging problems to be solved in milk lipidomic analysis highlighted.