Breast milk samples and 24-hour food records were obtained from lactating mothers on day 1 (colostrum), day 14 (transitional milk) and day 42 (mature milk) from Hangzhou (n = 202), Lanzhou (n = 133) and Beijing (n = 142), China. Fatty acid methyl esters were prepared by standard methods, separated and quantified by gas chromatography. We aimed to investigate the fatty acid composition (% of total fatty acid) in human milk of three lactating stages from three regions in China and the relationship with maternal dietary intake during lactation. Present results showed that the fatty acid composition of breast milk varied with lactating period and geographical regions in China. In all the milk samples, the total saturated fatty acid (SFA) remained stable. However, C10:0 and C12:0 increased over the lactation period, total monounsaturated fatty acid (MUFA) significantly increased from colostrum (34.50%) to transitional milk (37.06%), and total polyunsaturated fatty acid (PUFA) showed its highest percentage in colostrum (29.58%). In particular, C22:6n-3 and C22:5n-3 were lowest in mature milk (0.38% and 0.41%, respectively), and C18:3n-3 (1.83%) was lowest in colostrum. There were significant differences among the three regions in total MUFA and PUFA in breast milk. The Hangzhou samples had the lowest C18:1n-9 and highest C22:6n-3. Additionally, C22:6n-3, total PUFA and n-3 PUFA were lowest in the Lanzhou samples. Different dietary habits were largely the drivers behind the different fatty acid profiles among the three regions.