The vertebrate digestive tract, including that of humans, is the habitat to trillions of bacteria that are of significant importance to host biology and health. Although these communities are often postulated to have coevolved with their hosts, evidence is lacking, yet critical for our understanding of microbial symbiosis in vertebrates. To gain insight into the evolution of a gut symbiont, we have characterized the population genetic structure and phylogeny of Lactobacillus reuteri strains isolated from six different host species (human, mouse, rat, pig, chicken and turkey) using Amplified-Fragment Length Polymorphism (AFLP) and Multi-Locus Sequence Analysis (MLSA). The results revealed considerable genetic heterogeneity within the L. reuteri population and distinct monophyletic clades reflecting host origin but not provenance. The evolutionary patterns detected indicate a long-term association of L. reuteri lineages with particular vertebrate species and host-driven diversification. Results from a competition experiment in a gnotobiotic mouse model revealed that rodent isolates showed elevated ecological performance, indicating that evolution of L. reuteri lineages was adaptive. These findings provide evidence that some vertebrate gut microbes are not promiscuous, but have diversified into host-adapted lineages by a long-term evolutionary process, allowing the development of a highly specialized symbiosis.