Abstract Understanding how biological species arise is critical for understanding the evolution of life on Earth. Bioinformatic analyses have recently revealed that viruses, like multicellular life, form reproductively isolated biological species. Viruses are known to share high rates of genetic exchange, so how do they evolve genetic isolation? Here we evaluate two related bacteriophages and describe three factors that limit genetic exchange between them: 1) A nucleus-like compartment that physically separates replicating phage genomes, thereby limiting inter-phage recombination during co-infection. 2) A tubulin-based spindle that orchestrates phage replication and forms nonfunctional hybrid polymers. 3) A nuclear incompatibility factor that reduces phage fitness. Together these traits maintain species differences through “Subcellular Genetic Isolation” where viral genomes are physically separated during co-infection, and “Virogenesis Incompatibility” in which the interaction of cross-species components interferes with virus production.