Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) was applied for detailed characterization of fatty acid profile of 8 vegetable oils. Due to enhanced selectivity and sensitivity characteristics, the GC×GC method yielded more reliable quantification results compared to one dimensional gas chromatography, especially for medium-chain fatty acids and odd-carbon number fatty acids, which are present only at trace level. All problematic positional counterparts of unsaturated fatty acids (e.g. C21:0–C20:3 ω6, C20:3ω3–C20:4 ω6 and C20:5 ω3–C22:0), which commonly coeluted in the case of 1D gas chromatography, were baseline resolved. The specific compounds were found for particular vegetable oils, such as γ-linolenic acid for hempseed oil, heneicosylic acid and tricosylic acid for olive pomace oil, and nervonic acid for mustard oil.