The effect of spatial factors reflecting dispersal potential between sites versus local environmental conditions on freshwater planktonic communities remains poorly understood. We assessed differences in the relative importance of local and seasonal environmental conditions versus spatial factors in explaining differences in community composition (i.e., beta-diversity patterns) in microbial plankton of 39 mid-latitude Chilean lakes spanning representative ecological gradients in altitude, mixing depth and water chemistry. The assemblages were taxonomically profiled by paired-end high throughput sequencing of the V3-V4 region of the 16S and the V4 region of the 18S rRNA genes. Variation partitioning analyses revealed that the explanatory power of environmental and seasonal factors versus spatial variables and their mutual overlap varied considerably among taxa and functional groups. More than 12 % of the variation in community structure was uniquely explained by environmental factors in the phytoplankton groups Dinophyta, Ochrophyta and Cyanobacteria, as well as in oligotrophic ultramicrobacteria, such as small rhodopsin containing Actinobacteria and LD12 Alphaproteobacteria. In phago-heterotrophic and saprotrophic groups, including heterotrophic micro-eukaryotes, and Bacteroidetes, environmental factors explained a smaller or even insignificant portion of the differences in the community structure. Our findings suggest that in Chilean lake microplankton, complex traits related to ecological and trophic strategy appear to affect the relative effect of local environmental properties on their community composition and hence the strength of species sorting along limnological gradients.