Plant roots are important contributors of organic carbon compounds for soil organic matter (SOM) formation, particularly hemicellulose, cellulose and lignin, but little is known about the composition of many species. Knowledge of compositional changes as plant roots decompose is also limited. This information is essential to understand the role of root-derived macromolecules in SOM dynamics and carbon sequestration. The paucity of available data necessitates analytical techniques to assess root composition and changes during decomposition. The objectives of this research were to evaluate diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) for assessment of root cell wall composition and to quantify and characterize changes in the cell wall composition of important crop and forage species during decomposition in 30 and 270 day incubations. Results indicate that the roots of the same species are similar despite differences in climate, soil and fertilization, while important differences were noted between roots of different species. Spectral analysis was consistent with chemical fiber analysis composition data and revealed features that may be indicative of root suberin content. Between Day 0 and Day 30 significant (P<0.05) changes in alfalfa root hemicellulose, cellulose and lignin were observed as roots became enriched with lignin relative to hemicellulose and cellulose. No changes were observed in the other studied roots over this interval. In the 270 day incubation large species dependent variations were observed in the extent of root tissue decomposition. In contrast to the short term results, lignin, cellulose and hemicellulose in the roots of all studied species degraded proportionally over time. Analysis by DRIFTS supported the fiber analysis results and revealed important changes as roots decompose. Spectra illustrated changes in hemicellulose structure and potential suberin preservation in decomposing roots. These results help to increase understanding and prediction of soil organic matter dynamics which will help to predict possible impact of management changes or soil disturbance on soil health and productivity as well as long term organic carbon stabilization and the potential for C sequestration. Variability in root composition and degradation suggest that characterization of a range of individual species is necessary to predict the soil carbon contribution from roots.