Abstract: In order to evaluate the fatty acid composition characteristics of brown spotted grouper, a comprehensive analysis of the lipid yields, fatty acid compositions of total lipid and different lipid classes, and the stereospecific distributions of fatty acids in triacylglycerols (TAG) and phospholipids (PL) in muscle, head, skin and viscera was performed using solid-phase extraction and gas chromatography (GC) techniques. Rich saccharolipids (SL) and PL were found in the head and viscera. In the total fat of each tissue, the content of essential fatty acids ranged from 24.17% to 24.51% of the total fatty acids, and the content of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) ranged from 12.42% to 13.73% of the total fatty acids, with the index of atherogenicity, index of thrombogenicity and n-6/n-3 ratio were at low levels. Moreover, polyunsaturated fatty acids (PUFA), especially DHA+EPA (28.43%~37.37%), accounted for a much higher percentage of PL than in other lipid classes. Saturated fatty acids were mainly distributed in the sn-2 positions of TAG and PL, and monounsaturated fatty acids were mainly distributed in sn-1,3 positions of TAG and sn-2 position of PL. Furthermore, PUFA tended to be distributed in sn-1,3 positions of TAG and sn-1 position of PL. Interesting, DHA was mainly bonded to the sn-2 site of TAG and sn-1 site of PL, which was beneficial for their bioavailability. Hence, the lipid profiles of brown spotted grouper are abundant, and the positional distribution characteristics of DHA in TAG and PL makes it easier to be utilized by human body, which can be a potential source of DHA-TAG and DHA-PL with high comprehensive utilization value.