The aim of this study was to compare a molecular and a microscopic approach to study the planktonic eukaryotic diversity of an oligotrophic lake. Plankton samples from the temperate Lake Stechlin were assessed in winter and summer 2008 by comparison of 18S rRNA gene clone libraries to light microscopic evaluations. For both approaches identical samples were used. There were remarkable differences between the main groups recovered by the contrasting methods. The microscopic analyses showed predominance of autotrophic planktonic organisms, whereas most of them could not be discovered by the molecular method which resulted in a higher diversity of heterotrophic flagellates. The microscopic survey revealed high diversity of Chlorophyta and Cryptophyta as well as the Stramenopiles groups of Bacillariophyceae and Chrysophyceae. The clone libraries, based on full-length 18S rRNA gene sequences, displayed highest diversity of Alveolata belonging to seven different subclades. Notably, Antarctic Dinophyta-related clones were detected. The occurrence of the marine phagotrophic flagellate Telonema was also documented. Comparing the two sampling seasons, rich diversity suggests that flagellates played an important role in late winter (February), however, there is relatively low diversity in summer (August). The newly discovered molecular diversity of planktonic eukaryotes in Stechlin will help to understand the biodiversity patterns in freshwater lakes.