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Hemicelluloses (HC) are the second most abundant plant polysaccharides after cellulose, constituting 25-30% of plant materials. In spite of their abundance, HC are not effectively utilized. Recently, considerable interest has been directed to HC-based biomaterials because of their high oxygen barrier properties, which has potential in food packaging applications. In this study, HC were extracted from sugarcane bagasse and southern yellow pine using a hydrothermal technique which utilizes hot compressed water without catalyst. The parameters affecting the yield of extracted HC such as temperature, time and pressure, were tested and optimized. Eighty four percent of xylose was extracted from sugarcane bagasse at the optimum condition, 180 °C 30 min and 1 MPa pressure. In the case of southern yellow pine, 79% of the mannose was extracted at 190 °C for 10 min and 2 MPa pressure. Concentration and isolation of HC from bagasse and southern yellow pine HC extract were performed by membrane filtration and freeze drying systems. Isolated HC were characterized by FT-IR and 13C NMR techniques and used as a starting material for film preparation. Films were prepared in 0/100, 50/50, 60/40, 70/30 and 80/20% ratios of HC and sodium carboxymethylcellulose (CMC). Thirty five percent of sorbitol (w/w of HC and CMC weight) was also added as a plasticizer. Films were evaluated by measuring water absorption, water vapor permeability (WVP), tensile property and oxygen barrier capability. At 55% relative humidity (RH) and 25 °C the water absorption of both sugarcane bagasse and southern yellow pine HC-based films tended to increase as HC content increased. The lowest WVP of sugarcane bagasse (3.84e-12 g/Pa h m) and southern yellow pine HC films (2.18e-12 g/Pa h m) were determined in 60/40 HC/CMC films. Tensile test results showed that as HC content increases the Young’s modulus decreases, deflection at maximum load and percentage of strain at break increase. It implies that the film properties are changing from stiff to elastic. The oxygen permeability for 60/40 bagasse HC/CMC film was 0.005265 cc μm / (m2 day kPa) and for 70/30 pine HC/CMC film was 0.007570 cc μm /(m2 day kPa).