Despite their obvious importance, our knowledge about the eukaryotic microbial diversity of inland waters is still limited and poorly documented. We applied 18S rDNA amplicon sequencing to provide a comprehensive analysis of eukaryotic diversity in 74 low-productivity lakes along a 750 km longitudinal transect (5.40-18.52°E) across southern Scandinavia. We detected a wide diversity of pelagic microbial eukaryotes, classified into 1882 operational taxonomic units (OTUs). The highest OTU richness was found in traditional phytoplankton groups like Dinoflagellata, Chrysophyceae, Chlorophyta and Cryptophyta. A total of 53.6% OTUs were primarily autotrophic, while 19.4% of the heterotrophic OTUs belonged to putative parasitic taxa. Except for a longitudinal trend in the relative influence of mixotrophs, there were no significant associations between major functional groups (autotrophs, heterotrophs and parasites) and spatial and environmental variables. Community dissimilarity increased significantly with increasing geographical distance between lakes. In accordance with earlier, microscopy-based surveys in this region, we demonstrate distinct gradients in protistan diversity and community composition, which are better explained by spatial structure than local environment. The strong association between longitude and protistan diversity is probably better explained by differences in regional species pools due to differences in landscape productivity than by dispersal limitation or climatic constraints.