In recent years, two new approaches have been introduced in genetic studies of phytoplankton species. One is the application of highly polymorphic microsatellite markers, which allow detailed population genetic studies; the other is the development of methods that enable the direct genetic characterization of single cells as an alternative to clonal cultures. The aim of this study was to combine these two approaches in a method that would allow microsatellite genotyping of single phytoplankton cells, providing a novel tool for high-resolution population genetic studies. The dinoflagellate species Lingulodinium polyedrum (F. Stein) J. D. Dodge was selected as a model organism to develop this novel approach. The method we describe here is based on several key developments: (i) a simple and efficient DNA extraction method for single cells, (ii) the characterization of microsatellite markers for L. polyedrum, (iii) a protocol for the species identification of single cells through the analysis of partial rRNA gene sequences, and (iv) a two-step multiplex PCR protocol for the simultaneous amplification of microsatellite markers and partial rRNA gene sequences from single cells. Our protocol allowed the amplification of up to six microsatellite loci together with either the complete ITS1-5.8S-ITS2 region or a partial 18S region of the ribosomal gene of L. polyedrum from single motile cells and resting cysts. This article describes and evaluates the developed approach and discusses its significance for population genetic studies of L. polyedrum and other phytoplankton species.