Heterocystous filamentous cyanobacteria are regarded as the main N2-fixing organisms (diazotrophs) in the Baltic Sea. However, some studies indicate that picoplankton may also be important. The aim of this study was to examine the composition of putative diazotrophs in the picoplankton (< 3 µm) and to identify links to environmental factors. Nitrogenase (nifH) genes were amplified from community DNA by nested PCR, followed by cloning and sequencing. Clone libraries from nine environmental samples collected from the central Baltic Sea (April–October 2003, 3 m depth) and a negative control yielded a total of 433 sequences with an average clone library coverage of 92%. The sequences fell within nifH Clusters I, II and III and formed 15 distinct groups (> 96% amino acid similarity). Most of the sequences (77%) fell into nifH Cluster I (cyanobacteria and α-, β- and γ-Proteobacteria). However, only 26 sequences were related to cyanobacteria (e.g. Pseudanabaena) and among these no unicellular phylotypes were found. Sequences clustering with alternative nitrogenases (anfH) and Archaea were found in one sample while sequences related to anaerobic phylotypes were found in six samples distributed throughout the season. The identified phylogenetic groups showed covariance with several environmental factors but no strong links could be established. This suggests a variable and complex regulation of diazotrophic groups within Baltic Sea picoplankton.