Hemicellulose is an abundant and underutilized carbohydrate polymer in plants. The objective of this study was to understand the effect of delignification on hemicellulose extraction efficiency with different types of lignocellulosic biomass. In the case of pine, with a prior sodium chlorite or peracetic acid delignification, more than 50% of the original hemicellulose in the biomass could be extracted using a 10% sodium hydroxide solution; without delignification, only 3.4% of hemicellulose could be extracted from pine. In contrast, without prior delignification, acceptable hemicellulose extraction efficiencies (55.5% and 50.7%, respectively) were achieved from switchgrass and poplar. In addition, the effect of hemicellulose extraction processes on the enzymatic convertibility of the cellulose-rich residues after extraction was determined. The cellulose-rich residues from switchgrass after hemicellulose alkali extraction showed high glucose recovery with enzyme hydrolysis with or without prior delignification. For pine and poplar, high glucose recovery with enzyme hydrolysis of the cellulose-rich residues only occurred if the sample had a delignification step prior to hemicellulose extraction. This information on commercially available biomass feedstocks is useful for those considering isolating hemicellulose within a biorefinery concept.