Abstract: To investigate the difference characteristics of triacylglycerols (TAGs) in Xinjiang donkey milk, this study was based on the lipid metabolism concept of 'fatty acid (FA) composition-TAGs molecular structure', analyzed samples collected at 30, 60, and 90 days postpartum from three representative regions in Xinjiang: Wushi County (WS), Yiwu County (YW), and Kuche City (KC). Using gas chromatography (GC) and ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), combined with multivariate statistical methods such as principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), targeted lipidomic analysis of fatty acids (FAs) and TAGs was conducted. The study first clarified the composition characteristics of donkey milk fatty acids (FA), and then delved into the molecular differences of the TAGs constituted by them. In total, Yiwu donkey milk showed the lowest saturated FA/unsaturated FA ratio (0.59±0.01), a higher monounsaturated fatty acid content. Regarding lactation stages, SFA content was the highest at 30 days postpartum, dominated by palmitic acid. At 60 days, PUFA content peaked, with linoleic acid being the most abundant. Long-chain FAs (LCFA) predominated across all lactation stages (93.23%~93.83%), while medium-chain FAs (MCFA) and short-chain FAs (SCFA) represented a smaller proportion. Multivariate statistical analysis indicated, significant differences in TAG composition were observed across regions and lactation stages. In YW milk, TAGs enriched in unsaturated FAs were significantly upregulated, with C16:0 preferentially esterified at the sn-2 position. Comparisons between KC and YW revealed higher levels of medium- and short-chain saturated TAGs in KC milk. Additionally, TAG expression changed dynamically with lactation. Saturated TAGs and TAGs featuring C16:0 at the sn-2 position showed the lowest levels in the YW region after 60 days. From 60 to 90 days, long-chain TAG expression gradually increased. Significantly positive correlations were observed among TAG molecules with similar FA structures, chain lengths, or saturation levels, accompanied by specific negative correlations. From a lipidomics perspective, this study elucidates regional specificity and dynamic changes in FA and TAG composition in Xinjiang donkey milk throughout lactation. These findings provide a solid theoretical foundation and data evidence for the precise evaluation of donkey milk lipid quality, geographic origin identification, and the development of high-value-added dairy products.