Abstract: The position distribution of fatty acids in triglycerides from different parts of freshwater fish was investigated in this paper. The position distribution of fatty acids on triglycerides Sn-1,2,3 in four parts (head, tail, back, and viscera) from three freshwater fish species (grass, black, and crucian carp) was analyzed by gas chromatography after the solid phase extraction, enzymatic hydrolysis. The results showed that a total of 21 fatty acids were monitored on triglycerides of three freshwater fish species. The content of monounsaturated fatty acids (MUFAs) ranged from 29.58% to 49.48%, and MUFAs located in Sn-1,3 positions were significantly higher than those in the Sn-2 position in all samples (P<0.05), except for viscera of grass and black carp. The content of polyunsaturated fatty acids (PUFAs) varied from 17.51% to 46.55%, and PUFAs located in the Sn-2 position were significantly higher than those in Sn-1,3 positions in all samples (P<0.05), except for back and tail of grass carp and head and viscera of black carp. In the three kinds of fish, arachidonic acid (ARA) was evenly distributed in three positions of triglycerides. Eicosapentaenoic acid (EPA) was mainly distributed in the Sn-1,3 positions. In comparison, docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) were distributed primarily in the Sn-2 position. It can conclude that the fatty acid composition in different parts of different freshwater fish species was various, and the distribution of the same fatty acids on triglycerides in the varied elements of freshwater fish species was also different.