Lignocellulosic biofuels have been identified as a possible solution to contribute to the world’s demands in energy and environmental sustainability. However, the fundamental understanding of the physical and chemical traits hindering key reactions during biomass to biofuel conversion processes has been limited by the lack of suitable tools and by the large natural variability in such systems. Reaction wood constitutes a good model system to study variations of cellulose content, given the increase in cellulose content in the cell walls of the region under tension in the plant during growth. In this work, we use confocal Raman mapping and Pulsed Force Mode Atomic Force Microscopy (PFM) to explore the effect of variation in cellulose content on the structure and composition of the plant cell wall at the nanoscale. Using statistical analysis on Raman datasets, the characteristic peaks for cellulose and lignin are examined to reveal changes in peak positions across the different scanned regions of the cross section. PFM is used to study local mechanical properties of the different layers of the cell wall. Our approach facilitates the correlation of structure-composition traits of the plant cell wall for a more fundamental understanding of processes involved in biofuel research.