B-family DNA-directed DNA polymerases are DNA replication enzymes found in Eukaryota, Archaea, large DNA viruses, and in some, but not all, bacteria. Several polymerase domains are conserved among the B-family DNA polymerases from these organisms, suggesting that the B-family DNA polymerases evolved from a common ancestor. Eukaryotes retain at least three replicative B-family DNA polymerases, DNA polymerase α, δ, and ε, and one translesion B-family DNA polymerase, DNA polymerase ζ. Here, we present molecular evolutionary evidence that suggests DNA polymerase genes evolved through horizontal gene transfer between the viral and archaeal-eukaryotic lineages. Molecular phylogenetic analyses of the B-family DNA polymerases from nucleo-cytoplasmic large DNA viruses (NCLDVs), eukaryotes, and archaea suggest that different NCLDV lineages such as Poxviridae and Mimiviridae were involved in the evolution of different DNA polymerases (pol-α-, δ-, ε-, and ζ-like genes) in archaeal-eukaryotic cell lineages, putatively through horizontal gene transfer. These results support existing theories that link the evolution of NCLDVs and the origin of the eukaryotic nucleus.