Maternal fat intake and adipose reserves are major sources of PUFA during lactation. The present study examined the cross-sectional relationship between prolonged breast-feeding and maternal BMI, assessed adequacy of fat intake among lactating and non-lactating mothers of children 24–48 months of age and determined breast-milk fatty acid composition. Multi-stage sampling was used to select a representative sample of mothers from two rural districts in Bangladesh (n 474). Dietary data were collected during two non-consecutive 24 h periods via 12 h in-home daytime observations and recall. The National Cancer Institute method for episodically consumed foods was used to estimate usual intake distributions. Breast milk samples were collected from ninety-eight women, and breast-milk fatty acid methyl esters were quantified using GC. Approximately 42 % of lactating v. 26 % of non-lactating mothers were underweight (BMI < 18·5 kg/m2; P = 0·0003). The maternal diet was low in total fat (approximately 8 % of mean total energy) and food sources of PUFA, including oil and animal source foods, resulting in a low estimated mean total consumption of PUFA (5·1 g/d). Almost all women were estimated to consume less than the recommended intake levels for total fat, total PUFA, α-linolenic acid (ALA) and DHA. Median breast-milk linoleic acid (8·5 % weight) and ALA (0·2 %) concentrations were among the lowest reported in the literature, in contrast with arachidonic acid (0·5 %) and DHA (0·3 %) concentrations, which were mid-range. Bangladeshi women in general, and especially those who practise prolonged breast-feeding, may benefit from increased consumption of food sources of PUFA.