Cosmopolitan bacteria are those that are found practically everywhere in the world. One of them is Bacillus subtilis, which can travel around the world through dust storms rising from various deserts. Upon landing, bacterial survival is determined by the ability to adjust to the heterogonous environments and bacteria isolated from extremely different environments, such as desert and riverbank soil, are expected to be less related due to the environmental pressure of each region. However, little is known about the influence of soil and habitat on B. subtilis evolution. Here, we show that desert and riverbank B. subtilis strains differ in genetic relatedness and physiological traits, such as biofilm morphology and utilisation of carbon sources. Desert strains showed more diversity at the genetic level and were able to utilise more carbon sources than riverbank strains which were highly genetically conserved. Biofilm morphologies of desert and riverbank strains generally segregated and both groups formed different morphology clusters despite the astonishing diversity observed among riverbank strains. We also show that relatedness of B. subtilis strains does not decrease with distance inside the same habitat, which, together with diversity data implies that the difference in environmental selection pressures plays a fundamental role in the evolution of this species.