In this study, we aimed to detect the comprehensive fatty acid (FA) profile in human milk, including total fatty acids (total FAs), Sn-2 fatty acids (Sn-2 FAs), and phospholipid fatty acids (PLFAs), and further analyzed the influence of the sampling regions on human milk FA profile. The results showed that oleic acid (C18:1n9c), palmitic acid (C16:0), and linoleic acid (C18:2n6c) were the main FA species in total FAs, and Sn-2 C16:0 was the dominant Sn-2 FA species. Moreover, the PLFAs were mainly comprised of PL C16:0, PL stearic acid (PL C18:0), PL C18:1n9c, and PL C18:2n6c. Furthermore, FAs showed geographical differences, such as the highest content of linolenic acid (C18:3n3) and docosahexaenoic acid (C22:6n3) of total FAs was observed in Lanzhou and Weihai, respectively. Therefore, these results provide more FA data for the Chinese human milk database, and further enrich the theoretical information for the development of infant formula.