The composition and regiospecific distribution of fatty acids in TAG molecules is species-specific and varies within species. These are very important in determining milk fat's nutritional properties, impact on tissue metabolism, and functional properties affecting human health. TAG was hydrolysed by pancreatic lipase, and fatty acids were isolated by TLC separation and finally analysed using GC. Different trends were observed for the saturated and unsaturated fatty acid profiles among different species and within species, and the nutritional indices also showed significant differences between cow and buffalo milk fat. Strong species-wise variation in the sn-2 and sn-1(3) fatty acid distribution was often observed by analyzing data from across the whole year (intra-positional distribution). The mol % of saturated fatty acids at sn-2 positions was significantly (p≤0.05) higher in cow milk fat (73.49±0.53), while it was significantly (p≤0.05) higher in the sn-1(3) position in buffalo milk fat (71.23±1.61). On the contrary, the mol% of unsaturated fatty acids (UFAs) was significantly (p≤0.05) higher in the sn-2 position in buffalo milk fat (33.17±1.16) than in cow milk fat (26.51±0.84); the mol% of UFAs was significantly (p≤0.05) higher in the sn-1(3) position in cow milk fat (31.14±0.1) than in buffalo milk fat (25.33±0.59). A PCA analysis indicated that the sn-2-positional fatty acids were unique to each species. The MUFA and conjugated linoleic acid content were highest in the sn-2 position in buffalo milk. In buffalo milk fat, a higher amount of SFAs were converted to MUFA and PUFA, representing a higher Δ-9 desaturase index at the sn-2 position in TAG. PCA analysis also revealed differences in the sn-2- and sn-1(3)-positional distribution of TAG fatty acids in milk fat among different species. The Euclidean distance matrix of the quantitative data was also employed to group and categorize the fatty acids using heatmaps. The observed variations among various species revealed the significance of positional analysis in the characterization of milk fat from multiple species.