The evolutionary history and ecological differentiation of the genus Exiguobacterium was characterized within natural communities from the Cuatro Cienegas Basin, Mexico. Exiguobacterium comprises both halophilic and alkaliphilic bacteria that are abundant among the aquatic systems of the Cuatro Cienegas Basin. We obtained complete sequences of the 16srRNA gene and partial sequences of four housekeeping genes (citC, rpoB, recA and hsp70) in 183 Exiguobacterium isolates retrieved from distinct aquatic systems. We defined three main phylogroups that are closely related to marine and thermophilic species of the genus. These phylogroups were neither specific to a given aquatic system nor to a particular salinity. Phylogenetic reconstruction indicated the presence of several small clusters within the phylogroups. These clusters consisted of isolates predominantly retrieved from sediment or water. Unifrac and AdaptML analyses confirmed this observation, pointing towards a clear pattern of differentiation linked to either sediment or water habitats. Our results are in line with the concept that niche differentiation is one of the main factors shaping prokaryotic populations and leading to evolutionary divergence.