Freshwater aquifers in granitic rocks are widespread microbial habitats in the terrestrial subsurface. Microbial populations in deep granitic groundwater from two recently drilled (1 and 2 years) and two old boreholes (14 and 25 years) were compared. The 16S rRNA gene sequences related to "Candidatus Magnetobacterium bavaricum", Thermodesulfovibrio spp. of Nitrospirae (90.5-93.1 % similarity) and a novel candidate division with <90 % similarity to known cultivated species were dominant in all boreholes. Most of the environmental clones closely related to the novel lineages in Nitrospirae, which have been detected exclusively in deep groundwater samples. In contrast, betaproteobacterial sequences related to the family Rhodocyclaceae were obtained only from the recently drilled boreholes, which had higher total cell numbers. Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) analysis supported the result from clone library analysis; betaproteobacterial cells were dominantly detected in recently drilled boreholes. These results suggest that while indigenous microbial populations represented by the novel phylotypes persisted in the boreholes for 25 years, betaproteobacterial species disappeared after 2 years owing to the change of substrate availability.