In an effort to better understand and overcome biomass recalcitrance, six natural poplar variants were selected as the feedstock. Compositional analysis and physicochemical characterizations of these poplars were performed using different analytical techniques and the correlations between these physicochemical properties and enzymatic hydrolysis yield were investigated. Gel permeation chromatography (GPC) and 13C NMR were used to determine the degree of polymerization (DP) and crystallinity index (CrI) of cellulose, and the results along with the sugar release study indicated that cellulose DP likely played a more important role in enzymatic hydrolysis. Simons' stain revealed that accessible surface area of cellulose significantly varied among these variants from 17.3 to 33.2 mg/g biomass as reflected by dye adsorption, and cellulose accessibility was shown as one of the major factors governing substrates digestibility. HSQC and 31P NMR analysis detailed the structural features of isolated poplar lignin variants. Overall, cellulose relevant factors appeared to have a stronger correlation with glucose release, if any, than lignin structural features. Lignin structural features such as phenolic hydroxyl group and S/G ratio were found to have a more convincing impact on xylose release. Low lignin content, low cellulose DP and high cellulose accessibility generally favors enzymatic hydrolysis; however, recalcitrance cannot be simply judged on any solely substrate factor.