Viral infection of phytoplankton has major implications for biochemical and energy cycles, community dynamics, and microbial evolution in the marine environment. The non-bloom forming picoplankter Micromonas pusilla, a significant component of the plankton community worldwide, is known to be susceptible to infection by both dsDNA and dsRNA viruses. Logically, comprehensive knowledge of the ecology of M. pusilla requires a better understanding of the diversity and infection mechanisms of their viruses. Here, we investigated 19 new M. pusilla-specific viruses (MpVs) isolated from different locations and years. We performed partial characterization of those MpVs including structural characteristics, genome size, phylogenetic analysis based on partial DNA polymerase gene sequences, host range, and stability at different temperatures and upon exposure to chloroform. Combined, these characteristics allowed classification of the MpVs into 2 groups. Exposure to chloroform led to loss of infectivity by all MpVs in one group, which suggests the presence of an outer lipid envelope. In addition, all except one of the members in that group formed a monophylogenetic clade that was distinct from all other MpV isolates. The distinctive characteristics of the 2 MpV groups suggest different infection strategies, which may have important implications for the ecology of both host and virus populations in the environment. Knowledge gained from our study adds value to the MpV isolates as a scientific resource as it will aid in developing and testing in the laboratory new hypotheses about the ecological and biogeochemical implications of M. pusilla viral infection in the environment.