Bifidobacteria colonize the human gastrointestinal tract, vagina, oral cavity and breast milk. They influence human physiology and nutrition through health-promoting effects, play an important role as primary colonizers of the newborn gut, and contribute to vaginal microbiome homeostasis by producing lactic acid. Nevertheless, the mechanisms by which bifidobacteria are transmitted from mother to infant remains in discussion. Moreover, studies have suggested that Bifidobacterium spp. have specializations for gut colonization, but comparisons of strains of the same bifidobacteria species from different body sites are lacking. Here, our objective was to compare the genomes of Bifidobacterium breve (n = 17) and Bifidobacterium longum (n = 26) to assess whether gut and vaginal isolates of either species were distinguishable based on genome content. Comparison of the general genome features showed that vaginal and gut isolates did not differ in size, GC content, number of genes and CRISPR, either for B. breve or B. longum. Average nucleotide identity and whole genome phylogeny analysis revealed that vaginal and gut isolates did not cluster separately. Vaginal and gut isolates also had a similar COG (Cluster of Orthologous Group) category distribution. Differences in the accessory genomes between vaginal and gut strains were observed, but were not sufficient to distinguish isolates based on their origin. The results of this study support the hypothesis that the vaginal and gut microbiomes are colonized by a shared community of Bifidobacterium, and further emphasize the potential importance of the maternal vaginal microbiome as a source of infant gut microbiota.