<jats:title>Abstract</jats:title><jats:p>Lignocellulose, which includes mainly cellulose, hemicellulose and lignin, is a potential resource for the production of chemicals and for other applications. For effective production of materials derived from biomass, it is important to characterize the metabolites and polymeric components of the biomass. Nuclear magnetic resonance (NMR) spectroscopy has been used to identify biomass components; however, the NMR spectra of metabolites and lignocellulose components are ambiguously assigned in many cases due to overlapping chemical shift peaks. Using our <jats:sup>13</jats:sup>C-labeling technique in higher plants such as poplar samples, we demonstrated that overlapping peaks could be resolved by three-dimensional NMR experiments to more accurately assign chemical shifts compared with two-dimensional NMR measurements. Metabolites of the <jats:sup>13</jats:sup>C-poplar were measured by high-resolution magic angle spinning NMR spectroscopy, which allows sample analysis without solvent extraction, while lignocellulose components of the <jats:sup>13</jats:sup>C-poplar dissolved in dimethylsulfoxide/pyridine solvent were analyzed by solution-state NMR techniques. Using these methods, we were able to unambiguously assign chemical shifts of small and macromolecular components in <jats:sup>13</jats:sup>C-poplar samples. Furthermore, using samples of less than 5 mg, we could differentiate between two kinds of genes that were overexpressed in poplar samples, which produced clearly modified plant cell wall components.</jats:p>