Four different pretreatment technologies were applied to two Populus natural variants and the effects of each pretreatment on glucose release were compared. Physicochemical properties of pretreated biomass were analyzed by attenuated total reflection Fourier transform infrared spectroscopy, gel permeation chromatography, and cross polarization/magic angle spinning carbon-13 nuclear magnetic resonance techniques. The results revealed that hemicellulose and lignin were removed to different extents during various pretreatments. The degree of polymerization of cellulose was decreased in the order of alkali > hydrothermal > organosolv > dilute acid pretreatment. Cellulose crystallinity index was slightly increased after each pretreatment. The results also demonstrated that organosolv pretreatment resulted in the highest glucose yield. Among the tested properties of Populus, degree of polymerization of cellulose was negatively correlated with glucose release, whereas hemicellulose and lignin removal, and cellulose accessibility were positively associated with glucose release from the two Populus natural variants.