Phylogenetic relationships among picocyanobacteria from the Syn/Pro clade sensu Sánchez-Baracaldo et al. (2005) were determined using small subunit (ssu) rDNA sequences from novel culture isolates together with environmental samples from the Baltic Sea and seven freshwater lakes. The picocyanobacterial community comprised members of previously identified clades and of two previously undescribed clades. The number of well-supported clades suggests that freshwater picocyanobacterial communities encompass much greater diversity than is found in marine systems. To allow the quantification of community structure and temporal succession, clade-specific ssu rDNA TaqMan assays were designed and implemented. These assays were used to assess picocyanobacterial community structure in two lakes over an annual cycle in 2003/4, and in a small number of Baltic Sea samples collected in July 2003. In the lake-water samples, picocyanobacteria were found to be scarce during most of the year, with members of each clade reaching their peak abundance over a relatively short period during the summer (June to September), although representatives of the Cyanobium clade also developed an autumn peak extending towards the end of October. All four freshwater clades were present in the Baltic Sea, but their distribution was patchy over relatively short spatial scales. The use of molecular tools for describing and quantifying community structures reveals previously unexplored complexity in the phytoplankton and will facilitate the development of a more sophisticated understanding of community dynamics at the base of the food chains in lakes.