The trans isomers of 5,8,11,14,17-eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA) methyl esters were prepared by isomerisation with paratoluenesulfinic acid (PTSA) in dioxane. The isomers were fractionated by silver ion liquid chromatography with baseline resolution between the isomers with different number of trans double bonds. The fractions were analysed by GC-MS and the gas chromatographic properties of the EPA and DHA isomers with one and two trans double bonds were investigated on BPX-70 and SP-2560 cyanopropyl stationary phases. Different temperature and pressure programs were applied to introduce variations in retention indices of the isomers. The retention indices of all the trans isomers showed a strong linear correlation to the retention indices of the equivalent all-cis isomer, but the slopes for corresponding linear regression lines varied with the number of trans double bonds in the molecule. The regression lines were used to predict optimal conditions for the separation of trans isomers from the corresponding all-cis isomers. For DHA on BPX-70, and for EPA on both columns, it was possible to find windows where isomers with one trans double bond can be resolved from the corresponding all-cis isomers with R(s) > 1.0. In general, BPX-70 seems to have a more suitable selectivity for the analysis of these isomers than SP-2560. Two-dimensional fatty acid retention indices (2D-FARI) were found to be suitable for identification of trans geometry in polyunsaturated fatty acids (PUFA). Although there were substantial overlaps in the range of retention times between the all-cis isomers and isomers with one and two trans double bonds, 2D-FARI separated the isomers into distinct groups according to the number of trans double bonds.