A new, two-dimensional thin-layer chromatographic system was established to provide good separation of the unsaponifiable fractions from the seed oils of three wild Sesamum species, [S. alatum, Thonn.; S. radiatum, Schum and Thonn.; and S. angustifolium, (Oliv.) Engl.] and of the cultivated S. indicum, L. The system utilizes silica gel plates and n-hexane/diethyl ether (7:3, v/v) and chloroform/diethyl ether (9:1, v/v) as mobile phases in the first and second directions, respectively. The system could be used for qualitative studies and as a preparative technique for subsequent quantitative gas chromatographic separations in chemotaxonomic and related studies on Sesamum spp.