Viruses strongly influence the ecology and evolution of their eukaryotic hosts in the marine environment, but little is known about their diversity and distribution. Prasinoviruses infect an abundant and widespread class of phytoplankton, the Mamiellophyceae, and thereby exert a specific and important role in microbial ecosystems. However, molecular tools to specifically identify this viral genus in environmental samples are still lacking. We developed two primer sets, designed for use with polymerase chain reactions and 454 pyrosequencing technologies, to target two conserved genes, encoding the DNA polymerase (PolB gene) and the major capsid protein (MCP gene). While only one copy of the PolB gene is present in Prasinovirus genomes, there are at least seven paralogs for MCP, the copy we named number 6 being shared with other eukaryotic alga-infecting viruses. Primer sets for PolB and MCP6 were thus designed and tested on 6 samples from the Tara Oceans project. The results suggest that the MCP6 amplicons show greater richness but that PolB gave a wider coverage of Prasinovirus diversity. As a consequence, we recommend use of the PolB primer set, which will certainly reveal exciting new insights about the diversity and distribution of prasinoviruses at the community scale.