Phospholipids (PLs), including sphingomyelin, play important roles in cell membrane integrity, neural and brain development, and inflammatory responses. They are found in tissues and biological fluids, including human breast milk. In this cross-sectional study using high performance liquid chromatography–mass spectrometry, the average total PL concentrations in Malaysian mothers' colostrum and transitional milk were determined as 331 and 266 mg L −1 , respectively. In mature milk, the average total PL concentrations were 170, 210 and 220 mg L −1 at 2, 6 and 12 months, respectively, with a strong correlation between the total PL concentration and the fat concentration ( p < 0.001). The dominant PL class in mature milk was sphingomyelin (36–38%), followed by phosphatidylethanolamine (27–37%). The average maternal serum PL concentrations were higher in the third trimester (2089 mg L −1 ) than in the second (1667 mg L −1 ), with phosphatidylcholine predominant at 66% and with sphingomyelin at 22–24% of total PLs. [ABSTRACT FROM AUTHOR]