Triacylglycerols (TAG) of two different refined fish oils from sardine and a mixture of tuna and sardine oil were separated by reverse phase high performance liquid chromatography (RP-HPLC) with a binary solvent gradient of acetone/acetonitrile. Different fractions were observed in the chromatogram and TAG species were tentatively identified by subsequent analysis of the fatty acid (FA) profile in each fraction by capillary Gas Chromatography (GC). Peak identities were assigned on the basis of a multiple linear regression analysis by using factors such as carbon number, number of double bonds, number of monounsaturated fatty acids (MUFA) and number of polyunsaturated fatty acids (PUFA) in the molecule as predictors for TAG retention time. A successful correlation was obtained between retention times and the equivalent carbon number (ECN) of triacylglycerols. Regiospecific analysis of fatty acids in the TAG has been conducted by ethanolysis of the fish oil by using an immobilized lipase. The subsequent separation of 2-monoacylglycerol (2-MAG) by TLC (thin layer chromatography) analysis showed that ethanolysis system is effective for analysis of FA composition at the 2-position in oils containing PUFA. Principal components analysis (PCA) has been also applied to establish correlations between the different fatty acids in the TAG.