Picoplanktonic prasinophytes are well represented in culture collections and marine samples. In order to better characterize this ecologically important group, we compared the phylogenetic diversity of picoplanktonic prasinophyte strains available at the Roscoff Culture Collection (RCC) and that of nuclear SSU rDNA sequences from environmental clone libraries obtained from oceanic and coastal ecosystems. Among the 570 strains avalaible, 91 belonged to prasinophytes, 65 were partially sequenced, and we obtained the entire SSU rDNA sequence for a selection of 14 strains. Within the 18 available environmental clone libraries, the prasinophytes accounted for 12% of the total number of clones retrieved (142 partial sequences in total), and we selected 9 clones to obtain entire SSU rDNA sequence. Using this approach, we obtained a subsequent genetic database that revealed the presence of seven independent lineages among prasinophytes, including a novel clade (clade VII). This new clade groups the genus Picocystis, two unidentified coccoid strains, and 4 environmental sequences. For each of these seven lineages, at least one representative is available in culture. The three picoplanktonic genera Ostreococcus, Micromonas, and Bathycoccus (order Mamiellales), were the best represented prasinophytes both in cultures and genetic libraries. SSU rDNA phylogenetic analyses suggest that the genus Bathycoccus forms a very homogeneous group. In contrast, the genera Micromonas and Ostreococcus turned out to be quite complex, consisting of three and four independent lineages, respectively. This report of the overall diversity of picoeukaryotic prasinophytes reveals a group of ecologically important and diverse marine microorganims that are well represented by isolated cultures.