Anaerobic dark fermentation biohydrogen production from xylose was investigated under mesophilic (35 degrees C) and thermophilic (55 degrees C) conditions at various initial pH (5.0-10.0) and substrate concentrations (2.5-12.5 g/L). In addition, the microbial community structure variations under different temperatures were analyzed. It was demonstrated that the maximum hydrogen yield (1.24 mol-H-2/mol-xylose) was obtained with substrate concentration of 7.5 g/L and initial cultivation, pH 7.0, at 35 degrees C, with butyrate, acetate, and ethanol as the major byproducts. The increase of Substrate concentration resulted in accumulation of volatile fatty acids (VFAs), especially propionate, and a decrease in final pH under mesophilic conditions. However, the hydrogen yield increased along with the increase of substrate concentration at 55 degrees C with butyrate and ethanol as the main metabolite. Stable pH of the system could be maintained even at high xylose concentration up to 12.5 due to a low level of VFAs accumulation. A lower hydrogen yield of 1.14 mol-H-2/mol-xylose was Obtained at thermophilic condition, while a stable operation condition could be achieved and maintained more easily. Polymerase chain reaction-denaturing, gradient gel electrophoresis (PCR-DGGE) analysis showed that microbial community structures of both systems were dominated with bacterial species related to Clostridium, while the thermophilic system had fewer hydrogen-producing microbial species than that at mesothermal condition.