The application of microbial catalysts to syngas from the gasification of lignocellulosic biomass is gaining interest. Acetogens, a group of anaerobic bacteria, can grow autotrophically on gaseous substrates such as hydrogen and carbon dioxide or syngas and produce acetate via the acetyl-CoA pathway. Here, we report the isolation from a soil sample of two thermophilic acetogen strains, Y72 and Y73, that are closely related to Moorella sp. HUC22-1 and M. thermoacetica ATCC39073. The optimal growth temperature and pH for the strains were 60 °C and 6.0-6.5. Uracil auxotrophy was induced in them by replacing the orotate monophosphate decarboxylase gene (pyrF) with the kanamycin resistant marker (kan(r)). The transformants were isolated by supplementation of the basal medium with 300 mg/L of kanamycin. The transformation efficiency of strains Y72 and Y73 was 20-fold higher than that of strain ATCC39073. Hence these strains are considered possible hosts for thermophilic syngas fermentation.