The recently proposed order Candidatus Thermoprofundales, currently containing only one family-level lineage Marine Benthic Group-D (MBG-D), is distributed in global subsurface ecosystems and ecologically important, but its diversity, evolution and metabolism remain largely unknown. Here we described two novel family-level specialized lineages in Ca. Thermoprofundales, JdFR-43 and HyVt, which are restricted to specific biotopes (primarily in marine hydrothermal vents and occasionally in oil reservoirs and hot springs) in contrast to the cosmopolitan lineage MBG-D. The comparative genomics revealed that the specialized lineages have streamlined genomes, higher GC contents, enriched genes associated with nucleotide biosynthesis, ribosome biogenesis and DNA repair and additional thermostable aminopeptidases, enabling them to adapt to high-temperature habitats such as marine hydrothermal vents, deep subsurface oil reservoirs and hot springs. On the contrary, the unique metabolic traits of the cosmopolitan MBG-D, motility, glycolysis, butanoate metabolism, secondary metabolites production and additional genes for specific peptides and carbohydrates degradation potentially enhance its response to environmental change. Substrate preference is found for most MAGs across all lineages with the ability to utilize both polysaccharides (chitin and starch) and proteinaceous substances, whereas JdFR-43 members from oil reservoirs can only utilize proteins. These results expand the diversity of Ca. Thermoprofundales significantly and further improve our understandings of the adaptations of Ca. Thermoprofundales to various environments.