Polar lipids (PLs) are a significant functional component of milk that are difficult to quantitate. A simple method for comprehensive identification and quantitative analysis of all essential PL species using bovine milk is described. The lipid fraction was extracted by a mix of chloroform and methanol and the extract was directly used for PL identification and quantification. PLs were separated by hydrophilic interaction liquid chromatography (HILIC) and detected by an Orbitrap mass analyser in positive mode. The structure of PLs was established or confirmed by tandem MS in both positive and negative modes. The method is sensitive (with a LOD for all PL classes ≤0.1 ng) and reproducible, enabling simultaneous quantification of 70 PL species within a run of 45 min. Application of this method to the quantification of PLs in 32 bovine milk samples revealed the relative abundance of different PL classes, significant variation of PL content between individual samples and the correlation between the major PL classes. The method provides a tool for investigating the variation and metabolism of important PL components in bovine and human milk and in diverse mammalian species.