The lipid profiles of raw, pasteurized and fermented cow milk and goat milk were investigated using lipidomics based on ultra-high performance liquid chromatography (UPLC) coupled with quadrupole tandem electrostatic field orbitrap mass spectrometry. A total of 27 lipid subclasses were detected in all samples, including 1607 lipid molecules. The results proved that pasteurization had no effect on the lipid profiles of cow milk or goat milk, while fermentation had a significant influence on the lipid profiles of cow milk and goat milk; in particular, it greatly reduced the content of hemolytic phospholipids. Totally 27 and 23 lipid molecules which could be used as potential biomarkers for the identification of pasteurized and fermented milk were selected. This study has provided an understanding of the lipid profile and dynamic changes in the fermentation process of milk, which can provide a molecular basis for understanding the effects of heat treatment and fermentation on milk fat during yogurt processing and be helpful to understand the quality and nutritional characteristics of yogurt products.