Fluorescence in situ hybridization (FISH) was employed to elucidate of mesophilic (35°C) and thermophilic (55°C) syntrophic substrate oxidizing anaerobes, individually. The whole microbe cell was visualized in a wet amount of each enrichment culture with substrate of benzoate, ethanol and propionate, separately. Targeted cells, which determined previously by 16S rRNA-cloning analysis as the dominant microbe, were hybridized in situ by employing the specific 16S rRNA-tergeted oligonucleotide probes. In situ hybridization with known syntrophic oligonucleotide probes reveal the clearly syntrophic cells hybridized as the dominant cell growth in culture, such as Desulfovibrio, Geobacter-like cells. For specific detection of the new identified syntrophic anaerobes-like cells, the specific oligonucleotide probes were designed. The new probes were designed for the new cluster of phylum Firmicutes, obtained from mesophilic benzoate and thermophilic ethanol culture, individually. Additionally, the probe for the new mesophilic syntrophic propionate-oxidizing anaerobe, was also designed. These study revealed the detection of the probe reacted cell as the dominant cell in culture.