Candidate phylum KSB1 is composed of uncultured bacteria and has been reported across various environments. However, the phylogeny and metabolic potential of KSB1 have not been studied comprehensively. In this study, phylogenomic analysis of KSB1 genomes from public databases and eleven metagenome-assembled genomes (MAGs) from marine and hydrothermal sediments revealed that those genomes were clustered into four clades. Isolation source and relative abundance of KSB1 genomes showed that clade I was particularly abundant in bioreactor sludge. Genes related to dissimilatory reduction of nitrate to ammonia (DNRA), the last step of denitrification converting nitrous oxide to nitrogen and assimilatory sulfur reduction were observed in the expanded genomes of clade I, which may due to horizontal gene transfer that frequently occurred in bioreactor. Annotation and metabolic reconstruction of clades II and IV showed flagellum assembly and chemotaxis genes in the genomes, which may indicate that exploration and sensing for nutrients and chemical gradients are critical for the two clades in deep-sea and hydrothermal sediment. Metabolic potentials of fatty acids and short-chain hydrocarbons utilization were predicted in clades I and IV of KSB1. Collectively, phylogenomic and metabolic analyses of KSB1 clades provide insight into their anaerobic heterotrophic lifestyle and differentiation in potential ecological roles.