Abstract:Terrestrial hot spring, a typical extreme habitat, is the natural lab for exploring co-evolution of life and geological environment. The progress of molecular biological technology sheds light on decoding the "microbial dark matter" in this special habitat. Some outstanding achievements have been gained, such as expanding thermophilic microbial diversity, exploring new metabolic pathways and addressing their ecological functions, by using the combination of omics-based, probe-based and isotope-based approaches. This comprehensive review summarizes the development of molecular biological methods that were commonly used in the study of geo-microbiology in hot springs and discusses recent application of omics-based, probe-based and isotope-based technologies in revealing the microbial diversity and functional roles in terrestrial hot springs. Finally, we prospected the future research in geo-microbiology of terrestrial geothermal environments.