ABSTRACTTo what extent are generalist species cohesive evolutionary units rather than a compilation of recently diverged lineages? We examine this question in the context of host specificity and geographic structure in the insect pathogen and nematode mutualistXenorhabdus bovienii. This bacterial species partners with multiple nematode species across two clades in the genusSteinernema. We sequenced the genomes of 42X. bovieniistrains isolated from four different nematode species and three field sites within a 240-km2region and compared them to globally available reference genomes. We hypothesized thatX. bovieniiwould comprise several host-specific lineages, such that bacterial and nematode phylogenies would be largely congruent. Alternatively, we hypothesized that spatial proximity might be a dominant signal, as increasing geographic distance might lower shared selective pressures and opportunities for gene flow. We found partial support for both hypotheses. Isolates clustered largely by nematode host species but did not strictly match the nematode phylogeny, indicating that shifts in symbiont associations across nematode species and clades have occurred. Furthermore, both genetic similarity and gene flow decreased with geographic distance across nematode species, suggesting differentiation and constraints on gene flow across both factors, although no absolute barriers to gene flow were observed across the regional isolates. Several genes associated with biotic interactions were found to be undergoing selective sweeps within this regional population. The interactions included several insect toxins and genes implicated in microbial competition. Thus, gene flow maintains cohesiveness across host associations in this symbiont and may facilitate adaptive responses to a multipartite selective environment.