Abstract Objectives To investigate the fatty acids composition in different lactation stages of human milk in China. Methods A large cross-sectional study was conducted and lactating women (n = 6481) within 0–330 days postpartum were recruited in China between 2011 and 2013. Human milk sub-samples (n = 1135) were randomly selected for fatty acids analysis in the original study. The sample size of colostrum, transitional milk, and mature milk were 160, 177, 798 respectively. Fats were hydrolyzed and fatty acids methyl esters were analyzed using gas chromatography. Results The content of oleic acid, the highest level fatty acid in human milk, was 36.63%, 34.53% and 35.12% of total fatty acids in colostrum, transitional milk, and mature milk, respectively in China. The content of palmitic acid was about 23.69%, 21.90% and 20.82% in colostrum, transitional milk, and mature milk, respectively. Saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids accounted for 36.99%, 39.14% and 23.49% of total fatty acids of mature milk, respectively. The content of linoleic acid and arachidonic acid was 20.47% and 0.54% in mature milk, respectively. The content of eicosapentaenoic acid (EPA) and docosahexenic acid (DHA) was 0.06% and 0.28% in mature milk, respectively. In addition to lactation stage, the fatty acids content differed significantly among various regions, nationalities, or mode of delivery. (i.e., Regions (city vs rural): oleic acid: 33.8% vs 36.4%; linoleic acid: 22.88% vs 17.21%; arachidonic acid:0.64%vs 0.59%; DHA: 0.42% vs 0.26%; EPA: 0.09% vs 0.04%. Nationality (Han vs Minority): oleic acid: 34.40% vs 36.59%; linoleic acid: 21.69% vs 16.54%; arachidonic acid:0.64%vs 0.57%; DHA: 0.38% vs 0.24%; EPA: 0.08% vs 0.04%.). Conclusions Oleic acid is the most abundant fatty acids in human milk of China. In addition to lactation stage, the fatty acid concentrations vary among different regions and nationalities in China. Funding Sources This study was supported by Beijing Natural Science Foundation (S160002), the National Key R&D Program of China (2017YFD0400601) and the National High Technology Research and Development Program of China (863 Program) (2010AA023004).