This study characterizes milk triacylglycerol (TAG) and polar lipid (PL) fractions from cows and goats fed various lipid supplements modulating milk fat content. Twelve Holstein cows and 12 Alpine goats, at 86 ± 24.9 and 61 ± 1.8 days in milk, respectively, are allocated to one of 4 groups to receive diets supplemented with either corn oil [5% dry matter intake (DMI)] plus wheat starch (COS), marine algae powder (MAP; 1.5% DMI) or hydrogenated palm oil (HPO; 3% DMI), or a no-added-lipid control diet (CTL), according to a 4 × 4 Latin square design with 28 d experimental periods. Milk TAG and PL contents are determined by liquid chromatography-mass spectrometry (LC-MS). Multivariate analysis and ANOVA demonstrate major between-species differences in diet effects. In cows, COS specifically increases TAG 54:3 and 54:4 associated with milk fat depression (MFD), and increases the sum of phosphatidylcholines (PC) and phosphatidylinositols (PI). In addition to causing a MFD, MAP diet increases long-chain polyunsaturated TAG in both species, with higher magnitude in cows than in goats, and decreases the sum of PI in goats. HPO increases TAG 52:1 and the sum of PI in cows, but not in goats.