Abstract: In order to compare the difference of the muscle fatty acids in fishes family, the order and the species between freshwater fish, the crude fat of the back muscles of 15 kinds of freshwater fish was extracted with chloroform-methanol (volume ratio=2:1). The composition of fatty acids in 15 kinds of freshwater fishes was analyzed by gas chromatography-mass spectrometry.The results showed that the crude fat content of 15 freshwater fishes was 3.76%~14.74%, and 24 kinds of fatty acids of 15 freshwater fishes were detected, including 8 kinds of saturated fatty acids (SFA), 5 monounsaturated fatty acids (MUFA) and 11 polyunsaturated fatty acids (PUFA). The fatty acids of different freshwater fishes were basically the same except for some differences.Their contents were significantly different from those order, family and genus of the freshwater fish. The content of polyunsaturated fatty acids was the highest, all above 35%.The total content of EPA and DHA in the crude fat of different fish species was different.The contents of DHA and EPA in the crude fat of the perch were the highest, and the total content reached 30%.However, the total content of EPA and DHA in the crude fat of loach was only 8.51%.Some species of fish could be used as a good diet for EPA and DHA, and had higher utilization and development value.