The T4 and RB69 DNA replicative polymerases are members of the B family and are highly similar. Both replicate DNA with high fidelity and employ the same mechanism that allows efficient switching of the primer terminus between the polymerase and exonuclease sites. Both polymerases have a beta hairpin loop (hereafter called the beta loop) in their exonuclease domains that plays an important role in active-site switching. The beta loop is involved in strand separation and is needed to stabilize partially strand-separated exonuclease complexes. In T4 DNA polymerase, modification of the beta-loop residue G255 to Ser confers a strong mutator phenotype in vivo due to a reduced ability to form editing complexes. Here, we describe the RB69 DNA polymerase mutant with the equivalent residue (G258) changed to Ser but showing only mild mutator activity in vivo. On the other hand, deletion of the tip of the RB69 beta loop confers a strong mutator phenotype in vivo. Based on detailed mutational spectral analyses, DNA binding activities, and coupled polymerase/exonuclease assays, we define the differences between the T4 and RB69 polymerases. We propose that their beta loops facilitate strand separation in both polymerases, while the residues that form the loop have low structural constraints.