Nestedness measures the degree to which species assemblages are proper subsets of larger assemblages within a metacommunity. We analyzed nestedness in 20 freshwater data sets, each for a single metacommunity within a drainage basin. First, we examined the degree of nestedness across local assemblages and identified major environmental correlates of nestedness at sites. Second, we examined whether nestedness showed concordance among trophic levels. Third, we examined whether nested and idiosyncratic species (i.e., species that have unpredictable gaps in their distribution) differed ecologically and showed dissimilar distribution patterns along geographical and environmental gradients. Degrees of nestedness, obtained using a metric by Almeida-Neto et al. (2008), ranged from 22.9 to 57.0, with 11 matrices showing significant nestedness. Lake bacteria had a higher degree of nestedness than did phyto- or zooplankton. The degree of nestedness was related mainly to water temperature, conductivity, and trophic status of the water, results suggesting that habitat quality may be an important predictor of nestedness, but was only weakly concordant among trophic levels within metacommunities. However, averaged across all metacommunities, nestedness values between phytoplankton and zooplankton and between phytoplankton and bacteria were positively correlated. Nested and idiosyncratic species did not differ in their spatial and environmental distribution patterns. Freshwater planktonic assemblages were characterized by a relatively low degree of nestedness. The low congruence in nestedness among trophic levels suggests that assemblages typically show independent gradients of nestedness. This result indicates that assemblages respond dissimilarly to driving forces, such as habitat quality, or that nestedness in freshwaters might be related to stochastic colonization. Conserving only the most diverse sites may not protect rare species because of the relatively low degree of nestedness in fresh waters.