The microorganisms in the subterranean high-temperature petroleum reservoir is a kind of precious resource, which play an important role in matter circulation and energy flow in oil reservoir ecosystem. Traditionally, culturing techniques were used to analyze them, but it was hard to understand the microbial community diversity thoroughly. Molecular methods had overcome the pure culture bottleneck, which was very useful to analyze the environmental microbial community. In recent years, uncultured-based study of oil reservoir microbiology progressed rapidly, which produced important effect on the development of petroleum microbiology and ecology. In this paper, the current knowledge about molecular microbial ecology in petroleum reservoirs was reviewed and some suggestions towards further research were given.