The emergence of antiviral-resistant cytomegalovirus (CMV) strains is a continuing clinical problem, with increased numbers of immunocompromised patients given longer-duration antiviral prophylaxis. Two previously unrecognized CMV DNA polymerase mutations (N408K and A834P) identified separately and together in at-risk lung and kidney transplant recipients and a third mutation (L737M) identified in a liver transplant recipient were characterized by marker transfer to antiviral-sensitive laboratory strains AD169 and Towne. Subsequent phenotypic analyses of recombinant strains demonstrated the ability of mutation N408K to confer ganciclovir (GCV) and cidofovir (CDV) resistance and of mutation A834P to confer GCV, foscarnet, and CDV resistance. Mutation L737M did not confer resistance to any of the antiviral agents tested. A recombinant strain containing both N408K and A834P demonstrated increased GCV and CDV resistance compared to the levels of resistance of the virus containing only the A834P mutation. The addition of mutation N408K in combination with A834P also partially reconstituted the replication impairment of recombinant virus containing only A834P. This suggests that perturbation of both DNA polymerization (A834P) and exonuclease (N408K) activities contributes to antiviral resistance and altered replication kinetics in these mutant strains. The identification of these multidrug-resistant CMV strains in at-risk seronegative recipients of organs from seropositive donors suggests that improved prophylactic and treatment strategies are required. The additive effect of multiple mutations on antiviral susceptibility suggests that increasing antiviral-resistant phenotypes can result from different virus-antiviral interactions.