Among small photosynthetic eukaryotes that play a key role in oceanic food webs, picoplanktonic Mamiellophyceae such as Bathycoccus, Micromonas, and Ostreococcus are particularly important in coastal regions. By using a combination of cell sorting by flow cytometry, whole genome amplification (WGA), and 454 pyrosequencing, we obtained metagenomic data for two natural picophytoplankton populations from the coastal upwelling waters off central Chile. About 60% of the reads of each sample could be mapped to the genome of Bathycoccus strain from the Mediterranean Sea (RCC1105), representing a total of 9 Mbp (sample T142) and 13 Mbp (sample T149) of non-redundant Bathycoccus genome sequences. WGA did not amplify all regions uniformly, resulting in unequal coverage along a given chromosome and between chromosomes. The identity at the DNA level between the metagenomes and the cultured genome was very high (96.3% identical bases for the three larger chromosomes over a 360 kbp alignment). At least two to three different genotypes seemed to be present in each natural sample based on read mapping to Bathycoccus RCC1105 genome. Copyright: ß 2012 Vaulot et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the following programs: BIOSOPE cruise (Funded by the Centre National de la Recherche Scientifique), PICOFUNPAC (funded by the Agence Nationale de la Recherche, Biodiversité 06-BDIV-013), PHYTOMETAGENE (jointly funded by Japan Science and Technology and Centre National de la Recherche Scientifique), METAPICO (funded by Genoscope), Micro B3 (funded by the European Union, contract 287589) and FONDAP COPAS (funded by Comisió n Nacional de Investigació n Científica y Tecnoló gica, Gobierno de Chile). KV acknowledges the support of Ghent University (Multidisciplinary Research Partnership ''Bioinformatics: from nucleotides to networks '') and the Interuniversity Attraction Poles Programme [IUAP P6/25], initiated by the Belgian State, Science Policy Office (BioMaGNet). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.