Two milk samples were collected from 11 cows, one in the second week of lactation (8 to 14 days in milk; DIM) and one in late lactation (199 to 326 DIM) to assess the differences between early and late lactation in bovine milk fatty acid (FA) and triacylglycerol (TAG) composition, FA positional distribution in the TAG structure, and milk fat solid fat content (SFC). Cows in early lactation, that were in negative energy balance, mobilized C16:0, C18:0, and C18:1 cis 9 from their body fat stores, increasing the concentrations of these FA in milk fat. These high concentrations of C18:0 and C18:1 cis 9 enhanced the synthesis of high molecular weight TAG and decreased milk fat SFC in early lactation. For both lactation stages, alterations in the total FA concentrations in the TAG structures resulted in changes in the abundances and the proportions of the FA at the sn -2 and sn -1(3) positions in the TAG structure, yet without affecting the main esterification preferences of the FA.