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Freshwater bacterioplankton assemblages are composed of sympatric populations that can be delineated, for example, by ribosomal RNA gene relatedness and that differ in key ecophysiological properties. They may be free-living or attached, specialized for particular concentrations or subsets of substrates, or invest a variable amount of their resources in defence traits against protistan predators and viruses. Some may be motile and tactic whereas others are not, with far-reaching implications for their respective life styles and niche partitioning. The co-occurrence of competitors with overlapping growth requirements has profound consequences for the stability of community functions; it can to some extent be explained by habitat factors such as the microscale complexity and spatiotemporal variability of the lacustrine environments. On the other hand, the composition and diversity of freshwater microbial assemblages also reflects non-equilibrium states, dispersal and the stochasticity of community assembly processes. This review synoptically discusses the competition and niche separation of heterotrophic bacterial populations (defined at various levels of phylogenetic resolution) in the pelagic zone of inland surface waters from a variety of angles, focusing on habitat heterogeneity and the resulting biogeographic distribution patterns, the ecophysiological adaptations to the substrate field and the interactions of prokaryotes with predators and viruses.