The natural recalcitrance of biomass is due to the complex relationship between lignin-cellulose and hemicellulose in the plant cell wall.  Pretreatment technologies are required to reduce the recalcitrance of biomass prior to enzymatic deconstruction and one of the current challenges is to optimize this process while reducing the associated operating and capital costs. In this study, poplar was subjected to hot water pretreatment at 180 °C with treatment times ranging from 17 – 70 minutes. After pretreatment, the compositions of residual substrates were measured, and cellulose, hemicellulose, and lignin were isolated from the pretreated and starting poplar samples. Gel permeation chromatography and one- and two-dimensional nuclear magnetic resonance techniques were utilized to identify structural elements of importance to sugar release and recalcitrance of pretreated poplars. As pretreatment proceeded, cellulose was shown to decrease in chain length with  DP decreasing by 36% ~ 63% depending on pretreatment severity. A reduction in hemicellulose content was observed during poplar pretreatment and the residual hemicellulose had a decreased molecular weight. The crystallinity index of cellulose had no significant change after pretreatment and appeared unrelated to sugar release performance. Poplar lignin displayed a decrease in β-O-4 linkages but no significant change in methoxyl group content.Structural changes and their relationship to recalcitrance were examined from a perspective of future improvements in biomass pretreatment for enhanced sugar production for biofuels and lignin utilization.