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Metacommunity studies on lake bacterioplankton indicate the importance of environmental factors in structuring communities. Yet most of these studies cover relatively small spatial scales. We assessed the relative importance of environmental and spatial factors in shaping bacterioplankton communities across a >6000km latitudinal range, studying 48 shallow lowland lakes in the tropical, tropicali (isothermal subzone of the tropics) and tundra climate regions of South America using denaturing gradient gel electrophoresis. Bacterioplankton community composition (BCC) differed significantly across regions. Although a large fraction of the variation in BCC remained unexplained, the results supported a consistent significant contribution of local environmental variables and to a lesser extent spatial variables, irrespective of spatial scale. Upon correction for space, mainly biotic environmental factors significantly explained the variation in BCC. The abundance of pelagic cladocerans remained particularly significant, suggesting grazer effects on bacterioplankton communities in the studied lakes. These results confirm that bacterioplankton communities are predominantly structured by environmental factors, even over a large-scale latitudinal gradient (6026km), and stress the importance of including biotic variables in studies that aim to understand patterns in BCC.