Felled oil palm trunk (OPT) (25 years old) is an abundant biomass in Southern Thailand. The OPT composition was 31.28-42.85% cellulose, 19.73-25.56% hemicellulose, 10.74-18.47% lignin, 1.63-2.25% protein, 1.60-1.83% fat, 1.12-1.35% ash and trace amount of minerals (0.01-0.40%). Oil palm sap extracted from OPT was found to contain 15.72 g/L glucose, 2.25 g/L xylose, and 0.086 g/L arabinose. A total of twenty samples from hot springs (45-75 degrees C and pH 6.5-8.4), oil palm sap and palm oil mill effluent were enriched for isolation of hydrogen-producing bacteria. The highest hydrogen-producing strain was isolated from oil palm sap and identified as Clostridium beijerinckii PS-3 using biochemical test and 16S rRNA gene analysis. Among various carbon sources tested, glucose, xylose, starch and cellulose were the preferred substrates for hydrogen production. The strain PS-3 could produce the maximum hydrogen yield of 140.9 ml H-2/g total sugar and the cumulative hydrogen production of 1973 ml/L-oil palm sap. Therefore, C. beijerinckii PS-3 is a potential candidate for fermentative hydrogen production from mixed sugars of the oil palm sap. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.