Analyses of microbial communities from six water-flooding petroleum reservoirs at temperatures from 21 to 63 °C by 16S rRNA gene clone libraries indicates the presence of physiologically diverse and temperature-dependent microorganisms in these subterrestrial ecosystems. In samples originating from high-temperature petroleum reservoirs, most of the archaeal sequences belong to thermophiles affiliated with members of the genera Thermococcus, Methanothermobacter and the order Thermoplasmatales, whereas bacterial sequences predominantly belong to the phyla Firmicutes, Thermotogae and Thermodesulfobacteria. In contrast to high-temperature petroleum reservoirs, microorganisms belonging to the Proteobacteria, Methanobacteriales and Methanomicrobiales were the most encountered in samples collected from low-temperature petroleum reservoirs. Canonical correspondence analysis (CCA) revealed that temperature, mineralization, ionic type as well as volatile fatty acids showed correlation with the microbial community structures, in particular members of the Firmicutes and the genus Methanothermobacter showed positive correlation with temperature and the concentration of acetate. Overall, these data indicate the large occurrence of hydrogenotrophic methanogens in petroleum reservoirs and imply that acetate metabolism via syntrophic oxidation may represent the main methanogenic pathway in high-temperature petroleum reservoirs. © Author(s) 2012.