As a major component in plant cell walls, lignin is an important factor in numerous industrial processes, especially in wood saccharification and fermentation to biofuels. The ability to chemically differentiate and spatially locate lignins in wood cell structures provides an important contribution to the effort to improve these processes. The spatial distribution of the syringyl (S) and guaiacyl (G) lignins, both over larger regions and within a single cell wall, on poplar ( Populus trichocarpa ) wood cross-sections was determined via time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is the first time that direct chemically specific mass spectrometric mapping has been employed to elucidate the spatial distribution of S and G lignins. In agreement with results obtained by UV microscopy, ToF-SIMS images clearly show that the guaiacyl lignin is predominantly located in the vessel cell walls of poplar wood while syringyl lignin is mainly located in the fiber cell walls. The G/S ratio in vessel cell walls was determined to be approximately twice that found in fiber cell walls. A combination of Bi ToF-SIMS spectral image acquisition and C(60) sputtering provided the ability to attain the combination of spatial resolution and signal-to-noise necessary to determine the distribution of S and G lignins in a single cell wall. By this technique, it was possible to demonstrate that more guaiacyl lignin is located in the middle lamella layer and more syringyl lignin is located in the inner cell wall area.