Abstract:Objective Rummeliibacillus, a genus encompassing three known species, R. stabekisii, R. pycnus, and R. suwonensis, has a wide range of potential applications in biodegradation, probiotics, animal feed, and production of arginine, caproic acid, and other compounds. This study aims to explore the genetic diversity of this genus at the genomic level.Methods A comparative pangenome analysis of 12 strains isolated from different sources was conducted. In addition, the phylogenetic analysis, functional annotation, genomic metabolic pathway analysis, and prediction of mobile genetic elements were carried out.Results A total of 8 024 gene clusters were identified. The core genome, accessory genome, and strain-specific genes comprised 1 550, 3 941, and 2 533 gene clusters, respectively. In the core genome, the arginine cycle of six strains was complete. Seven strains had the ability to completely biosynthesize acetoin. However, only R. pycnus and R. suwonensis 3B-1 were able to completely biosynthesize caproic acid. The phylogenetic tree, DNA-DNA hybridization, and average nucleotide identity showed that Rummeliibacillus sp. G93 and Rummeliibacillus sp. TYF-LIM-RU47 were strains of R. stabekisii. Rummeliibacillus sp. POC4 and Rummeliibacillus sp. TYF005 may belong to a new species of this genus. In addition, genomic islands were identified in all the 12 strains, with the number ranging from four (R. stabekisii DSM 25578 and R. stabekisii NBRC 104870) to 14 (Rummeliibacillus sp. SL167 and Rummeliibacillus sp. TYF005), and prophage sequences were found in five of the 12 strains.Conclusion This study provides a genomic framework for Rummeliibacillus that could assist the further exploration of this genus.