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Clostridium tyrobutyricumis a promising microbial host for the anaerobic production of bio-based chemicals, especially butyric acid. However, the limited genetic traits of C. tyrobutyricumresulted in its constrained applicability in the food industry due to the absence of publicly available data. We performed a comparative genomic analysis of 13 C. tyrobutyricumstrains isolated from different habitats to investigate the adaptation mechanisms and metabolic potentials of the C. tyrobutyricumstrains isolated from fermentation environments (FE). The results showed that the genetic diversity of FE- associated C. tyrobutyricumstrains was higher than that of strains isolated from non-fermentation environments (NFE). Based on evaluating safety-related genes, FE-associated strains were likely non-pathogenic to humans or animals. Many environmental adaptation-related genes involved in energy production and conversion, phosphotransferase system (PTS), etc., were significantly enriched in FE-associated strains, which was conducive to their survival in the corresponding habitats. The analysis of carbohydrate-active enzymes revealed that the CAZyme categories of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) were found to be significantly enriched in FE-associated strains, which was beneficial for carbohydrate utilization (including starch, cellulose, etc.). Fermentation experiments revealed that strains isolated from the Chinese liquor fermentation environment were capable of decomposing carbohydrates and producing organic acids. Collectively, this study provided insights into the genomic features of C. tyrobutyricumand the theoretical basis for further use, research, and development of these strains.