In this work, aspen and wheat straw substrates were pretreated using aqueous dilute acid (DA) and chemimechanical (CM) pretreatment, and the impact of these pretreatments on biomass lignin was investigated. Here DA pretreatment refers to the acidic treatment of biomass powders and CM pretreatment refers to the acidic treatments of chips or stalks followed by disc refining. The resulting substrates were observed using advanced microscopy techniques to determine changes in lignin distribution throughout the fiber cell wall due to the acid treatments. Following acid treatment, lignin within the cell wall was divided into spherical or elongated bundles. The surface of DA and CM pretreated substrates were shown to differ, with CM pretreated aspen exhibited a more cellulose-rich surface. X-ray Photoelectron Spectroscopy data indicated reduced surface lignin on CM pretreated aspen and increased surface lignin on CM pretreated wheat straw. Fourier transform infrared spectroscopy (FT-IR) spectra illustrated changes in bands related to hemicellulose lignin, and surface hydroxyl content suggesting possible differences in lignin chemistry. It was hypothesized that differences in lignin distribution based on biomass type and pretreatment method were due to interactions between the chemical treatment and the mechanical size reductions steps. Particle size prior to chemical treatment could impact the movement of lignin onto sample surface, and chemical treatment prior to size reduction could impact the fracture plane during the downsizing process. It is expected that both these effects could impact lignin distribution within the sample and on the sample surface. (C) 2016 Published by Elsevier Ltd.