2-Hydroxy fatty acids (2-OH-FAs) and 3-hydroxy fatty acids (3-OH-FAs) were recently identified at trace levels in dairy products and other food samples (vegetable oils and animal brains). Due to the asymmetric carbon bearing the hydroxy group, they are chiral. This study focused on the enantioselective determination of 2- and 3-OH-FAs in food. For this purpose, extracted saponifiable lipids were converted into methyl esters, and the resulting fatty acid methyl esters (FAMEs) were separated into OH-FAMEs (minor fraction) and non-OH-FAMEs (bulk fraction). OH-FAMEs were then derivatized with (R)-(-)-alpha-methoxy-alpha-trifluoromethylphenylacetyl chloride [(R)-(-)-MTPA-Cl, Mosher's reagent] to produce the corresponding MTPA-O-FAMEs. MTPA-O-FAME diastereomers were then analyzed by gas chromatography with electron capture negative-ion mass spectrometry (GC/ECNI-MS) in the selected ion monitoring (SIM) mode. In the food samples, both (S)- and (R)-enantiomers of 2- and 3-OH-FAs were detected, with the (R)-enantiomer being enantiopure or predominant with one exception. Especially 2- and 3-OH-16:0 were found to contain relevant proportions of the (S)-enantiomer. The differences in enantiomeric composition of 2- and 3-OH-16:0 detected for cheese samples were proposed as markers for authenticity controls.