The aim of this study was to determine the fatty acid (FA) composition of breast milk, and its association with mothers’ FA intake. Milk samples were obtained from 238 healthy lactating women who volunteered to participate in the Human Milk Micronutrients Analysis Research. Dietary intake during lactation was assessed using a 3-d food record, and fat content and FA composition of the breast milk samples were analysed by IR spectrometry using MilkoScan FT2 and GC flame ionisation detector, respectively. The fat content was 3·31 (sd 1·41) g/100 ml breast milk. The concentrations of arachidonic acid (20 : 4 n-6), EPA (20 : 5 n-3) and DHA (22 : 6 n-3) in breast milk were 0·48 (sd 0·13), 0·15 (sd 0·12) and 0·67 (sd 0·47) % of total FA, respectively. Fat content and FA composition of breast milk were associated with maternal age, BMI, supplement use and infant age. Dietary intakes of EPA, DHA, n-3 FA, n-6 FA, SFA and PUFA were positively correlated with the corresponding FA in the milk samples. FA levels in breast milk and maternal diet are highly correlated. Further studies are warranted to explore factors that may be associated with changes in FA composition in human milk.