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A PCR-DGGE (denaturing gradient gel electrophoresis of polymerase chain reaction) protocol was used for monitoring the dynamic changes in the microbial population during photohydrogen production. Total DNA was extracted directly from the mixed bacterial community in the reactor and subjected to PCR with V3-16S rDNA and pufM gene primers, and the amplifications were then analyzed by DGGE. The DGGE patts demonstrated the dynamics of community structure and the shift of microbial diversity, which corresponded to different running periods of the reactor. The optimal hydrogen producing community formed on day 10. Using DGGE analysis with the pufM gene fragments was superior to V3-16S rDNA region genes for detecting the dynamic variations of the photosynthetic bacteria population during hydrogen production. The comparative sequence analysis of excised DGGE bands showed the relationship between specific population structures and system performance. Rhodopseudomonas palustris was presumed as one of the dominant community members for hydrogen production in the reactor. The PCR-DGGE protocol was proven to be a good tool for monitoring the photohydrogen production in real time and offered the available information to improve the photohydrogen producing system.