AbstractIt has been proposed that early bacteria, or even the last universal common ancestor of all cells, were thermophilic. However, research on the origin and evolution of thermophily is hampered by the difficulties associated with the isolation of deep-branching thermophilic microorganisms in pure culture. Here, we isolate a deep-branching thermophilic bacterium from a deep-sea hydrothermal vent, using a two-step cultivation strategy (“Subtraction-Suboptimal”, StS) designed to isolate rare organisms. The bacterium, which we name Zhurongbacter thermophilus 3DAC, is a sulfur-reducing heterotroph that is phylogenetically related to Coprothermobacterota and other thermophilic bacterial groups, forming a clade that seems to represent a major, early-diverging bacterial lineage. The ancestor of this clade might be a thermophilic, strictly anaerobic, motile, hydrogen-dependent, and mixotrophic bacterium. Thus, our study provides insights into the early evolution of thermophilic bacteria.