A high-affinity divalent cation-binding site located proximal to the catalytic center has been identified in several RNA-dependent RNA polymerases (RdRps), but the characteristics of such a site have not been systematically studied. Here, all available polymerase structures that follow the hand-like structural motif were screened for the presence of a divalent cation close to the catalytic site but distinct from catalytic metal ions. Such non-catalytic ions were found in all RNA virus families for which there were high-resolution RdRp structures available. Bound ions were always located in structurally similar locations at an approximate 6-Å distance from the catalytic site. Furthermore, the second aspartate residue in the highly conserved GDD sequence was found to be involved in the coordination of the bound ion in all viral RdRps studied. These results suggest that a non-catalytic ion-binding site is conserved across positive-sense, single-stranded, and double-stranded RNA viruses. Interestingly, a non-catalytic ion was also observed in a similar position in the reverse transcriptase of the human immunodeficiency virus. Moreover, two members of the DNA-dependent DNA polymerase B family displayed an ion at a comparable distance from the catalytic site, but the position was clearly distinct from the non-catalytic ion-binding sites of RdRps.