In this study， the fatty acid composition and content in human milk of different cities and lactational periods were analyzed. A total of 204 human milk samples were collected from Baotou (n=41)， Beijing (n=40)， Jinan (n=20)， Kunming (n=33)， Shenzhen (n=21) and Xining (n=49)， including colostrum (n=28)， one month postpartum (n=67)， and four month postpartum (n=109). The content of fatty acids in human milk was analyzed by gas chromatography using C11 ∶ 0 as the internal standard. This study investigated the effects of lactational period and geographical location on fatty acids content of human milk in China. The results showed that the fat content in mature human milk was significantly higher than that in colostrum (P<0.05)， the fat content in Jinan human milk was the highest (~5 g/100 mL)， and the lowest were Kunming and Shenzhen (<3.0 g/100 mL). The content of C16 ∶ 0 during lactation was relatively stable at around 192 mg/g fat. The colostrum was rich in long-chain unsaturated fatty acids， and the content of C18 ∶ 2 n-6c and C18 ∶ 3 n-3 in colostrum was significantly lower than that in mature human milk (P<0.05). There were certain differences in the fatty acid content of human milk in different regions， and the ratio of linoleic acid/linolenic acid varies greatly. Further comparisons were made between the data in this paper and the fatty acid content of human milk in other countries. This study provided theoretical support for the quantitative analysis of human milk fatty acids as well as the influencing factors of human milk fatty acids concentration.