A gas chromatographic (GC) method is described for the indirect enantioresolution of 2- and 3-hydroxy fatty acids (OH-FAs). It combines the derivatization of each alkylated enantiomer and the subsequent transfer with (R)-(-)-alpha-methoxy-alpha-trifluoromethylphenylacetyl chloride [(R)-(-)-MTPA-Cl, Mosher's reagent] into a diastereomeric (S)-MTPA derivative. The enantiomers of each derivatized OH-FA were well separated on three non-chiral GC-columns (CP-Sil 2, CP-Sil 8/20% C18 and VF-5ms). The derivatives were detected with high sensitivity by GC with electron-capture detection (GC/ECD) and electron-capture negative-ion mass spectrometry (GC/ECNI-MS) because of their enhanced electron-capturing properties. When applied to sunflower oil spiked with a small amount of a racemic 2-OH-FA, the present method allowed for a highly selective identification without influence from the sample matrix. For more complex samples such as wool wax, GC/ECNI-MS was superior to GC/ECD, since the high sensitivity of this method was linked with high selectivity. Using GC/ECNI-MS in the selected ion monitoring (SIM) mode, 16 enantiopure 2-OH-FAs were detected in a wool wax sample.