This study was conducted to demonstrate the application of quantitative real-time polymerase chain reaction (qRT-PCR) for the quantification of dominant bacteria in an anaerobic reactor using a designed TaqMan probe. A novel group of uncultured thermophilic bacteria affiliated with Thermotogales was first found in a phenol-degrading sludge from a 55°C upflow anaerobic sludge blanket (UASB) reactor, which effectively removed 99% of phenol at loading of 0.51 g-phenol l−1 d−1 h of hydraulic retention. A TaqMan probe was then designed targeting this group of Thermotogales affiliated bacteria (TAB), and used to monitor its concentration in the reactors. Results showed that the TAB population in the 55°C reactor increased proportional to the phenol degrading rate. Results also showed that the TAB population ranged 3.5–9.9% in the 55°C phenol-degrading sludge, but only 0.0044% in the 37°C sludge and 0.000086% in the 26°C sludge.