This work reports for the first time the use of on-line coupling of reversed phase liquid chromatography with gas chromatography for the stereodifferentiation of oak lactone, which is considered to be one of the main constituents of the aroma of different products. As the determination of its four possible stereoisomers is often difficult due to the many variables which influence the enantiomeric and diastereomeric separations in complex matrices, different multidimensional chromatographic techniques (i.e., on-line coupled reversed phase liquid chromatography with gas chromatography as well as multidimensional gas chromatography) are used in the present work to perform the stereochemical analysis of oak lactone (i.e., 5-n-butyl-4-methyl-4,5-dihydro-2(3H)-furanone). As a result, the four stereoisomers of oak lactone, namely (4S,5S) cis, (4S,5R) trans, (4R,5R) cis and (4R,5S) trans, can be eventually resolved in a commercial oak extract using either heptakis-(2,3,6-tri-O-methyl)-β-cyclodextrin or octakis-(3-O-butyryl-2,6-di-O-pentyl)-γ-cyclodextrin as chiral stationary phases.