During the enzymatic hydrolysis of the cellulose component of the overall lignocelluloses-to-bioethanol process, lignin has been shown to be very influential, acting as both a physical barrier and limiting hydrolysis. Although hydrophobic, electrostatic and hydrogen bonding interactions between the lignin and cellulases have been suggested to influence the hydrolysis efficiency, comparative studies using isolated lignins from different types of biomass which have been pretreatment in different ways have not been done. To gain a better understanding of the effects of lignin on enzymatic hydrolysis, six different substrates: steam and organosolv pretreated softwood (lodgepole pine), hardwood (poplar) and an agricultural residue (corn stover), were prepared and lignin was isolated from the pretreated substrates by two different enzyme and chemically based methods. The lower lignin yields obtained with corn stover when compared to poplar and lodgepole pine suggested that the hydrophobicity of the corn stover derived lignin was lower than the lignin from the poplar and lodgepole pine. Subsequent characterization of the physical and chemical properties of the isolated lignins showed that the carboxylic acid present in the isolated lignin had a significant influence on enzymatic hydrolysis yields when lignin was added to pure cellulose. Dehydrogenative polymers (DHP) from ferulic acid adsorbed lower amounts of cellulases and did not decrease hydrolysis yields when compared to the DHP from coniferyl alcohol. This indicated that the increased carboxylic acid content of the lignin at least partially alleviated the non-productive binding of cellulases to the substrate and increased the overall enzymatic hydrolysis of the cellulose.