Herein, polar lipids, including glycerophospholipid, lysophospholipid, cholesterol (CHOL), sphingomyelin (SM), and ceramide (Cer), in human breast milk were subjected to lipidomic analysis via UPSFC-Q-TOF-MS. A total of 11 lipid classes and 195 molecular species were identified and quantified in human breast milk samples (n = 126) from three lactational stages across three regions in China. CHOL and SM together account for more than 50 % of the total polar lipids. The total polar lipids, SM, and phosphatidylethanolamine (PE) were ∼ 54.5, ∼17.8, and ∼ 3.5 mg/100 mL, respectively, which both significantly (P <
0.05) increased from colostrum to later lactational stages. DHA is highly enriched (68 % of the total DHA) in PE molecules. SM and Cer contain high ratios of nervonic acid (24:1 n-9), ∼35 % and ∼ 23 %, respectively. General patterns exist in the variation of polar lipid molecular species in human breast milk across different regions throughout the lactational stages.