Hydrological and geological heterogeneity in the subsurface can isolate ground - water bodies in an aquifer system and create hydrologically distinct aquifers overlying each other with varying amounts of water exchange and unknown amounts of biological exchange. The hetero geneous nature of these subsurface waters likely drives changes in groundwater microbiological parameters. In the present study, flow cytometry was used to examine the abundance and cytometrically defined subpopulation structure of bacteria and virus-like particles (VLPs) in 3 distinct, vertically stratified aquifer layers consisting of an unconfined aquifer, a confining layer and a confined aquifer. Despite total microbial abundances remaining constant, the composition of bacterial and VLP communities varied among the aquifer layers. Cytometrically defined subpopulations were defined by nucleic acid content and size and ranged from 1 bacterial and VLP subpopulation in the unconfined aquifer to 4 bacterial and 3 VLP subpopulations in the confined aquifer. This variability in the subpopulation assemblages is likely driven by a combination of hydrological heterogeneity and biological interactions. The results presented here indicate complexity in microbial communities in discrete aquifer layers that may be overlooked when reporting general abundances. Groundwater bacteria and VLPs appear to be a sensitive indicator of the biological dynamics of aquifer systems and may be used to identify heterogeneous water bodies and help distinguish individual aquifer layers in an aquifer system.