Pseudo-lignin, which can be broadly defined as aromatic material that yields a positive Klason lignin value and is not derived from native lignin, has been recently reported to form during the dilute acid pretreatment of poplar holocellulose. To investigate the chemistry of pseudo-lignin formation, GPC, FT-IR and 13 C NMR were utilized to characterize pseudo-lignin extracted from dilute-acid pretreated α-cellulose and holocellulose. The results showed that pseudo-lignin consisting of carbonyl, carboxylic, aromatic and aliphatic structures was produced from dilute acid pretreated cellulose and hemicellulose. Pseudo-lignin extracted from holocellulose pretreated at different conditions had similar molecular weights (M n ∼1000g/mol; M w ∼5000g/mol) and structural features (carbonyl, carboxylic, aromatic and methoxy structures). These characterizations have provided the pseudo-lignin formation mechanisms during pretreatment. The presence and structure of pseudo-lignin is important since pseudo-lignin decreases the enzymatic conversion.