Abstract Understanding human milk triacylglycerols (TAGs) provides valuable insights for infant formula development. This study utilized ultra-performance supercritical fluid chromatography to investigate how lactation stages and maternal diet affect TAG profiles. 18:1/16:0/18:1 (O/P/O), 16:0/18:1/16:0 (P/O/P), and 18:1/16:0/18:2 (O/P/L) were most abundant in human milk, accounting for 20% of the total TAGs. Furthermore, significant differences in TAG compositions were observed across the three stages of lactation. Also, there are apparent differences in the TAG of Egyptian and Chinese human milk. Egyptian human milk exhibited higher levels of TAGs esterified with three saturated fatty acids (SSS), TAGs esterified with two saturated fatty acids, and one unsaturated fatty acid (S2U), and total palmitic acid compared to Chinese human milk. On the other hand, TAGs with three long-chain fatty acids (LLL) and TAGs with three unsaturated fatty acids (UUU) were at higher levels in Chinese human milk. Understanding the prevalence of these specific TAGs, particularly related to O/P/O, can help in formulating infant formulas that aim to mimic the composition of human milk more accurately.