Little is known about fatty acid (FA) distribution among triacylglyceride (TG), cholesterol ester (CE), and phospholipid (PL) plasma lipid fractions in lactating women, and the relationships between these fractions and the FA profile of the milk that mothers produce. Milk fat and serum lipid FA compositions in lactating women were compared to investigate which serum lipids are the source of FA in milk fat. A TLC methodology was used to fractionate serum lipids including PL subclasses, that is, phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM). Plasma lipids consisted of CE (36.4%), TG (19.1%), PE (1.40%), PC (39.80%), and SM (3.29%). With regard to PL fractions, PE was characterized by the highest levels of 18:0, 20:4 n‐6, 22:6 n‐3, and alkenyl ethers. PC revealed 16:0 as the predominant FA, followed by 18:2 n‐6, 18:0, cis‐9 18:1, and 20:4 n‐6, whereas saturated FA from 16:0 to 24:0 were characteristic of SM. Although n‐6 and n‐3 polyunsaturated FA were mostly found in PE and PC, strong correlations were found for 18:2 n‐6 and 18:3 n‐3 between plasma TG and milk lipids. This study also shows that trans 18:1 isomers may be selectively incorporated into milk fat from specific serum fractions.Practical applications:An easy‐to‐use and inexpensive two‐stage TLC methodology to fractionate plasma lipids requiring only 1 mL of blood serum has been developed. Moreover, this study provides valuable information on the composition of serum lipids in lactating women, particularly with regard to PL classes, and their influence on the milk fat FA profile.Fatty acid transfer from serum lipids to milk fat in lactating women.