The two-step dilute sulfuric acid pretreatment is frequently employed during the conversion of lignocellulosic biomass to bioethanol.  Chemical characterization of the solid material after pretreatment usually reveals a higher content of acid insoluble (Klason) lignin compared to the starting biomass.  This has been attributed to the formation of condensed lignin-like compounds from the degradation of carbohydrates under acidic conditions.  However, direct evidence has never been presented for this hypothesis.  In this study, holocellulose and hemicelluloses isolated from hybrid poplar have been subjected to the two-step dilute acid pretreatments at three different severities, and the resulting material analyzed using a variety of spectroscopic and chemical characterization techniques.  Starting with holocellulose which has a very low (1.6 %) Klason lignin content, the acid pretreated material shows an increased lignin content especially at higher severity.  At the highest severity, the pretreated holocellulose has 96 % Klason lignin.  Solid-state CP/MAS 13C NMR and FTIR spectra of the acid treated holocellulose and hemicelluloses show the appearance of carboxylic and unsaturated carbon signals.   This is accompanied by an increase in the carbon content and decreased oxygen content with increasing severity.  Extraction with acidic dioxane and subsequent solution NMR analysis reveals that lignin like material is indeed formed during acid pretreatment of the carbohydrate fraction of poplar.  Results from detailed characterization of the acid treated poplar carbohydrates will be presented and the impact of the formation of lignin-like compounds on biomass recalcitrance and enzymatic deconstruction will be reviewed.