High-resolution selected-ion recording (SIR) of the exact molecular ion mass was used to confirm unambiguously the presence of conjugated linoleic acid (CLA) derivatives in biological matrices and standard mixtures and to differentiate non-CLA derivatives from CLA derivatives in the CLA region of the gas chromatogram. The success of this method was based on the selectivity of the SIR technique and its sensitivity, which was comparable to that of flame-ionization detection. A minor fatty acid methyl ester (FAME) was identified as methyl heneicosanoate (21:0), and six isomers of 20:2 FAME were found to elute in the CLA region. Isomerization of a standard CLA mixture resulted in a non-CLA flame-ionization response eluting in the CLA region of the gas chromatogram. It is therefore recommended that the identification of minor CLA isomers in natural products or biological matrices should include their direct confirmation by mass spectrometry.