Most studies of mixed culture fermentation for hydrogen production focus on the metabolite distribution under different operational conditions. While, the corresponding microbial dynamics research is limited. The aim of this work was to reveal the microbial community dynamics of extreme-thermophilic mixed culture enriched in a chemostat for hydrogen production with the combining molecular tools of denaturing gradient gel electrophoresis (DGGE), Illumina Miseq high-throughput sequencing platform, and 16S rRNA clone library sequencing. The analyzed results showed that the genera of Caldanaerobius, Caldicellulosiruptor and/or Thermoanaerobacter dominated the bacteria. While archaea were not detected even by Illumina Miseq high-throughput sequencing. The effects of hydrogen partial pressure or pH increasing from 5.5 to 7.0 on the microbial communities were not notably, and genera of Caldicellulosiruptor and Thermoanaerobacter were the dominants. High hydrogen yield in this chemostat was attributed to the enrichment of Caldicellulosiruptor. While Caldanaerobius dominated the bacteria communities as pH decreased to 4.5. Caldanaerobius and Thermoanaerobacter dominated at high influent glucose concentrations. Thereby, it clearly indicated that the microbial dynamics shall be considered when analyzing the extreme-thermophilic mixed culture fermentation in the future. (c) 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.