In this study, we investigated effects of bacterial stress treatment on domination of hydrogen producing bacteria. As the bacterial stress treatment to control hydrogen fermentation microflora, we tested heat stress (105°C, 2 h) and freeze-thaw stress (a total of 3 cycles, each including-15°C, 12 h and 4°C, 6 h). The effect of bacterial stress on glucose hydrogen fermentation characteristic was determined by batch test using glass bottles. The results of the batch cultivation indicated that hydrogen fermentation characteristics differ among the stressed microflora. This result suggests that each original seed source has the suitable bacterial stress treatment to increase the hydrogen production. At the same time, we conducted 16S rRNA gene based analysis of hydrogen fermentation microbial community by PCR-minigel-single-strand conformation polymorphism (PCR-minigel-SSCP) method. Clostridium species such as Clostridium butyricum were identified from hydrogen fermentation batch culture.