Organic matter preserved in cell walls of permineralized plant fossils was analyzed by using scanning transmission X-ray microscopy and spectroscopy at energies near the 1s absorption edge of carbon. Microchemical analyses were performed directly on cellulose acetate peels of the fossils, preserving information on the anatomical distribution of organic materials. Individual tracheid walls in both Eocene and Early Devonian fossils exhibit spatially distinct chemical zoning inherited from original wall biopolymers and cell-wall microstructure. Molecular analysis of submicrometer domains using carbon X-ray absorption near-edge spectroscopy documents the differential distribution of hydroxylated aromatic and alcohol (and/or ether) carbon in the inner and outer regions of tracheid walls. This zonation reflects the deposition of lignin and structural polysaccharides in Devonian plants, indicating biochemical and developmental pathways similar to those of living tracheophytes.