Milk fat triacylglycerols (TAGs) analysis is considered to be a challenging task; the considerable numbers of fatty acyl moieties form one of the most complicated natural fats, with potential thousands of molecular species of TAGs. Herein, TAGs fingerprinting of raw and processed bovine and buffalo milk was carried out using the ultra-performance liquid chromatography-mass spectrometry. Firstly, milk samples were, respectively, subjected to pasteurisation, homogenisation and freeze-drying, and the TAGs profile was then analysed. Palmitic, oleic, myristic and stearic were the predominant fatty acids detected. The results revealed that TAGs profile was not significantly influenced by the applied technological treatments. Bovine milk TAGs exhibited 28–54 total acyl carbons, with 0–5 double bonds at 80 different retention times. However, buffalo milk TAGs possessed 26–54 total acyl carbon number containing 0–4 double bonds at 84 different retention times. The findings of this study could offer a valuable knowledge to the field of dairy technology.