Hydrogen production by dark fermentation is a promising approach for sustainable energy production. However, the effect of substrate pretreatment and microbial community dynamics on hydrogen yield remains an area requiring further research. This study aimed to investigate the changes in microbial profiles in a hydrogen-producing bioreactor with three different operating parameters. Furthermore, [FeFe]-hydrogenase, an enzyme notoriously difficult to detect in mixed cultures, was characterized according to the dominant species in the reactors. In the bioreactors, differences in the hydrogen-producing microbial popu-lation were monitored, focusing on the effects of particle size and heat pretreatment of fruit and vegetable waste. These microbial differences were analyzed using next-generation sequencing. Species-specific primers were designed to target the hydA gene of [FeFe]-hydro-genase in Clostridium butyricum in mixed cultures. Phylogenetic relationships of [FeFe]-hydrogenase partial amino acid sequences were evaluated. The results showed that members of the genera Clostridium and Lactobacillus predominated in thermally pretreated waste reactors with higher hydrogen yields. C. butyricum (46.93%) was predominant in reactors containing waste particles larger than 5 cm, while Lactobacillus fermentum (28.28%) was the most abundant species in reactors with waste particles smaller than 5 cm. Phylogenetic analysis revealed that the [FeFe]-hydrogenase of C. butyricum showed close similarity to those of Clostridium saccharobutyricum, Clostridium sp., and Clostridium chromiireducens. These findings provide new insights into hydrogen-producing microbial dynamics under different operating conditions and support the use of species-specific hydA gene characterization in mixed cultures.