AbstractThe deep biosphere is an energy constrained ecosystem yet fosters diverse microbial communities that are key in biogeochemical cycling. Whether microbial communities in deep biosphere groundwaters are shaped by infiltration of allochthonous surface microorganisms or the evolution of autochthonous species remains unresolved. In this study, 16S rRNA gene amplicon analyses showed that few groups of surface microbes infiltrated deep biosphere groundwaters at the Äspö Hard Rock Laboratory, Sweden, but that such populations constituted up to 49% of the microbial abundance. The dominant persisting phyla included Patescibacteria, Proteobacteria, and Epsilonbacteraeota. Despite the hydrological connection of the Baltic Sea with the studied groundwaters, infiltrating microbes predominantly originated from deep soil groundwater. Most deep biosphere groundwater populations lacked surface representatives, suggesting that they have evolved from ancient autochthonous populations. We propose that deep biosphere groundwater communities in the Fennoscandian Shield consist of selected infiltrated and indigenous populations adapted to the prevailing conditions.