The genetic diversity of picoplanktonic (i.e. cells that can pass through a 3 μm pore-size filter) green algae was investigated in the Mediterranean Sea in late summer by a culture-independent approach. Genetic libraries of the 18S rRNA gene were constructed using two different primer sets. The first set is commonly used to amplify the majority of eukaryotic lineages, while the second was composed of a general eukaryotic forward primer and a reverse primer biased towards the phylum Chloroplastida. A total of 3980 partial environmental sequences were obtained: 1668 using the general eukaryotic primer set and 2312 using the Chloroplastida-biased primer set. Of these sequences, 65 (4%) and 594 (26%) belonged to the Chloroplastida respectively. A 99.5% sequence similarity cut-off value allowed classification of these 659 Chloroplastida sequences into 74 different operational taxonomic units. A majority of the Chloroplastida sequences (99%) belonged to the prasinophytes. In addition to the seven independent prasinophyte lineages previously described, we discovered two new clades (clades VIII and IX), as well as a significant genetic diversity at the species and subspecies levels, notably among the genera Crustomastix, Dolichomastix and Mamiella (Mamiellales), but also within Pyramimonas and Halosphaera (Pyramimonadales). Such diversity within prasinophytes has not previously been observed by cloning approaches, illustrating the power of using targeted primers for clone library construction. Prasinophyte assemblages differed especially in relation to nutrient levels. Micromonas and Ostreococcus were mainly recovered from mesotrophic areas, whereas Mamiella, Crustomastix and Dolichomastix were mostly detected in oligotrophic surface waters. Within genera such as Ostreococcus or Crustomastix for which several clades were observed, depth seemed to be the main factor controlling differential distribution of genotypes.