Unicellular green picophytoplankton from the Mamiellales order are pervasive in marine ecosystems and susceptible to infections by prasinoviruses, large double-stranded DNA viruses within the Nucleocytoviricota phylum. We developed a double-stranded DNA virus enrichment and shotgun sequencing method, and successfully assembled 80 prasinovirus genomes from 43 samples in the South China Sea. Our research delivered the first direct estimation of 94% accuracy in correlating genome similarity to host range. Stirkingly, our analyses uncovered unexpected host-switching across diverse algal lineages, challenging the existing paradigms of host–virus co-speciation and revealing the dynamic nature of viral evolution. We also detected six instances of horizontal gene transfer between prasinoviruses and their hosts, including a novel alternative oxidase. Additionally, diversifying selection on a major capsid protein suggests an ongoing co-evolutionary arms race. These insights not only expand our understanding of prasinovirus genomic diversity but also highlight the intricate evolutionary mechanisms driving their ecological success and shaping broader virus–host interactions in marine environments.