Lactobacilli are dominant members of the “healthy” female urogenital microbiota. One of these species, Lactobacillus jensenii, is routinely identified in the urinary microbiota of women both with and without urinary tract symptoms. In March 2020, the new bacterial species Lactobacillus mulieris was introduced, and phylogenetic and average nucleotide identity analysis identified eight L. jensenii strains that should be classified as members of the L. mulieris species. This prompted our phylogenomic study of all publicly available L. jensenii and L. mulieris genome sequences. While there is little variation in the 16S rRNA gene sequences, the core genome shows a clear distinction between genomes of the two species. We find eight additional strains of the species L. mulieris among these genomes. Furthermore, one strain, currently classified as L. mulieris UMB7784, is distinct from both L. jensenii and L. mulieris strains. As part of our comparative genomic study, we also investigated the genetic content that distinguishes these two species. Unique to the L. jensenii genomes are several genes related to catabolism of disaccharides. In contrast, L. mulieris genomes encode several cell surface and secreted proteins that are not found within the L. jensenii genomes. These L. jensenii-specific and L. mulieris-specific loci provide insight into phenotypic differences of these two species.