Molecular imaging of separate but still incompletely resolved spots on high-performance thin-layer chromatography (HPTLC) plates is used for the direct analysis of porcine brain lipids by desorption electrospray ionization mass spectrometry (DESI-MS). Eight class-specific spots were imaged in the negative ion mode and shown to contain more than fifty lipids. A low lateral resolution of 400 x 400 microm allowed simple, rapid, and incomplete separation to be combined with DESI imaging for the identification of many components of these extremely complex mixtures. In this work, tandem mass spectrometry (MS/MS) was also employed to confirm the identity of particular lipids directly on HPTLC plates.