Microbial fermentation is a promising technology for hydrogen (H-2) production. H-2 producers in marine geothermal environments are thermophilic and halotolerant. However, no one has surveyed an environment specifically for thermophilic bacteria that produce H-2 through Fe-Fe hydrogenases (H(2)ase). Using heterotrophic medium, several microflora from a seaweed bed associated with marine hot springs were enriched and analyzed for H-2 production. A H-2-producing microflora was obtained from Sargassum sp., 16S rRNA genes and Fe-Fe H(2)ase diversities of this enrichment were also analyzed. Based on 16S rRNA genes analysis, 10 phylotypes were found in the H-2-producing microflora showing 90.0-99.5 % identities to known species, and belonged to Clostridia, Gammaproteobacteria, and Bacillales. Clostridia were the most abundant group, and three Clostridia phylotypes were most related to known H-2 producers such as Anaerovorax odorimutans (94.0 % identity), Clostridium papyrosolvens (98.4 % identity), and Clostridium tepidiprofundi (93.1 % identity). For Fe-Fe H(2)ases, seven phylotypes were obtained, showing 63-97 % identities to known Fe-Fe H(2)ases, and fell into four distinct clusters. Phylotypes HW55-3 and HM55-1 belonged to thermophilic and salt-tolerant H-2-producing Clostridia, Halothermothrix orenii-like Fe-Fe H(2)ases (80 % identity), and cellulolytic H-2-producing Clostridia, C. papyrosolvens-like Fe-Fe H(2)ases (97 % identity), respectively. The results of both 16S rRNA genes and Fe-Fe H(2)ases surveys suggested that the thermophilic and halotolerant H-2-producing microflora in seaweed bed of hot spring area represented previously unknown H-2 producers, and have potential application for H-2 production.