Yak milk is a promising lipid source substitute for infant formulas designed to mimic human milk. However, comparative studies on the lipid profiles between human and yak milk are scarce. To address this gap, in this study, we thoroughly analysed and compared the lipidome and fatty acid (FA) composition of human colostrum, human mature milk and yak mature milk. A total of 2686 lipid species from 30 lipid classes were identified in the three milk types. Notably, yak mature milk surpassed both human milk stages in the total content of lipid species, triglycerides (TG) and saturated FA. In particular, three potential lipid biomarkers, namely TG(6,0_8,0_14:0) + NH4, TG(16,0_6,0_8:0) + NH4 and TG(10,0_12,0_12,0) + NH4, were identified to differentiate yak mature milk from human colostrum and mature milk. Moreover, upon analysing the lipid metabolic pathways, it was found that the lipids involved in the pathways of acetylcholine synthesis, as well as starch and sucrose metabolism, may not manifest notable differences between yak mature milk and human colostrum, indicating the presence of similar neurodevelopment-regulating and metabolic characteristics in yak milk as in colostrum. Therefore, this comprehensive comparison offers novel insights into the potential of yak mature milk lipids to enhance the humanisation of infant formulas.