The components and contents of fatty acids in the muscle and the hepatopancreas of Penaeus vannamei cultured in the ponds of sea- and fresh-waters were determined. Lipid was extracted from tissues with "Folch" solvent under ultrasonication, and esterified with KOH-BF3 method followed by capillary gas chromatography. A fused-silica BPX70 capillary column (60 m x 0.25 mm i.d.) and flame ionization detector (FID) were employed. Fatty acids, more importantly, the positional isomers, were identified by applying equivalent chain length (ECLT) values based on even-chain saturated fatty acid methyl ester standards. Area normalization method can be used to quantify the contents of the fatty acids. The results indicated that between the two kinds of shrimps the nature of major fatty acids were the same, but there were more differences in the contents of major fatty acids. In all the tested fatty acids, the unsaturated fatty acids (UFA) were majority and the contents of polyunsaturated fatty acids were obviously higher than monounsaturated fatty acids. In the muscle lipid of the two kinds of shrimps, the total contents of eicosapentaenoic acid (EPA) and docosahexenoic acid (DHA) (highly unsaturated fatty acids) were very high, which reached to over 20%. In the hepatopancreas, the total contents of EPA and DHA were lower (ranged from 11% to 17%). Contents of n-6 cluster UFA in fresh-water shrimps were higher than that in sea-water shrimps. The value of (n-3)/(n-6) of sea-water shrimps was higher than that of fresh-water shrimps.