Comprehensive two-dimensional gas chromatography (GC x GC) is a recent technique, rapidly gaining importance for the analysis of complex samples. Here, we evaluate the potential of GC x GC for the analysis of the fatty acid profile of milk from dairy cows fed either a control diet or the control diet supplemented with marine algae. Fatty acids were separated using two column combinations, a nonpolar/polar arrangement and a polar/nonpolar arrangement. Due to the difference in separation mechanism of the two columns, GC x GC resulted in an improved separation compared with analysis on the same column set without the use of the modulator. Displaying the peaks in a 2-D contour plot showed a well-ordered structure of fatty acids according to their number of carbon atoms and degree of unsaturation, facilitating identification of known and unknown compounds. Based on these relations, identification of carbon number and degree of unsaturation of several 22-fatty acids was possible. The large difference between the 22-fatty acids from milk fat of cows fed the control and the marine algae-containing diet suggest that rumen hydrogenation of 22:6 n-3 results in a similar complex profile of hydrogenation intermediates as observed for 18:2 n-6 and 18:3 n-3. In conclusion, this experiment suggests GC x GC to be a powerful technique for the analysis of fatty acids. Nevertheless, further research on the optimization of GC x GC is needed to increase separation of trans- and cis-18:1 isomers, which may require a longer and/or more selective 1 D column.