Viruses play an important role in the regulation of phytoplankton populations. In the Mediterranean Sea, prasinophyte green algae are abundant and widespread, and within this group the genera Bathycoccus, Micromonas and Ostreococcus (Mamiellales) are the most common. Although these organisms constitute a significant part of the marine ecosystem, little is known about the viruses infecting them. We showed that double-stranded DNA viruses, likely members of the Phycodnaviridae family, can infect and grow in different host laboratory prasinophyte strains. Different pairs of degenerate primers were designed to PCR amplify a region of the conserved viral polymerase gene in order to characterize these viral strains. Twenty-seven new viral strains from five different host strains were thus analysed. We established phylogenetic trees for the hosts (18S) and their associated viruses (partial polymerase gene) and discuss the taxonomic significance of Phycodnaviridae. Within eukaryotic double-stranded DNA viruses, we showed that viruses from Bathycoccus, Micromonas and Ostreococcus form a monophyletic group that we refer to as ‘Prasinovirus’.