A protocol for the analysis of the positional distribution of fatty acids (FA) in solid triacylglycerols (TAG) was developed using sn-1(3) selective alcoholysis catalyzed by immobilized Candida antarctica lipase B (CALB). One part by weight of solid fat and ten parts by weight of ethanol (99.5 %) were warmed to liquefy the fat. After adding 0.44 parts by weight of CALB, the mixture was shaken at 50°C for 10 min then at 30°C for 2.8 h. The recovery of 2-MAG after the 3-h transesterification reaction was ca. 85 % of the maximum theoretical yield (33 mol%), with the loss of 15 % attributable to the acyl migration from sn-2 to sn-1(3). The recovery was similar to that of the solvent-free alcoholysis of structured lipids, 1,3dipalmitoyl, 2-oleoyl glycerol and 1,3-dioleoyl, 2-palmitoyl glycerol, conducted at 30°C for 3 h. In contrast, the acyl migration from sn-1(3) to sn-2 was hardly observed.Because the detected acyl migration was only in the direction of sn-2 to sn-1(3), and not vice versa, it is proposed to determine the FA composition of the sn-2 position of TAG by the gas chromatographic analysis of 2-MAG fraction recovered from the enzymatic reaction mixture, and the FA composition of sn-1(3) position by a mass balance using the FA composition of TAG and of the sn-2 position as inputs. The procedure was successfully applied to palm oil and shea butter, and docosahexaenoic acid (DHA)-rich single cell oil from Aurantiochytrium sp. KH105 for the first time.