Lactobacillus fermentum is a lactic acid bacterium frequently isolated from mammal tissues, milk, and plant material fermentations, such as sourdough. A comparative genomics analysis of 28 L. fermentum strains enabled the investigation of the core and accessory genes of this species. The core protein phylogenomic tree of the strains examined, consisting of five clades, did not exhibit clear clustering of strains based on isolation source, suggesting a free-living lifestyle. Based on the presence/absence of orthogroups, the largest clade, containing most of the human-related strains, was separated from the rest. The extended core genome included genes necessary for the heterolactic fermentation. Many traits were found to be strain-dependent, for instance utilisation of xylose and arabinose. Compared to other strains, the genome of L. fermentum IMDO 130101, a candidate starter culture strain capable of dominating sourdough fermentations, contained unique genes related to the metabolism of starch degradation products, which could be advantageous for growth in sourdough matrices. This study explained the traits that were previously demonstrated for L. fermentum IMDO 130101 at the genetic level and provided future avenues of research regarding L. fermentum strains isolated from sourdough.