In this paper, we report the use of mass spectrometry imaging and structural analysis of lipids directly on a tissue specimen, carried out by means of matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry, using a combination of spiral orbit-type and reflectron-type time-of-flight mass spectrometers. The most intense peak observed in the mass spectrum from a brain tissue specimen was confirmed as phosphatidylcholine (34 : 1) [M+K]+, using tandem mass spectrometry. The charge remote fragmentation channels, which are characteristically observed using high-energy collision induced dissociation, contributed significantly to this confirmation. Accurate mass analysis was further facilitated by mass correction using the confirmed peak. In mass spectrometry imaging, the high resolving power of our system could separate doublet peak of less than 0.1 u diveerence, which would otherwise be problematic when using a low-resolution reflectron type time-of-flight mass spectrometer. Two compounds, observed at m/z 848.56 and 848.65, were found to be located in complementary positions on a brain tissue specimen. These results demonstrate the importance of a high-performance tandem time-of-flight mass spectrometer for mass spectrometry imaging and analysis of observed compounds, to allow distinction between biological molecules.