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We report the case of a lung transplant recipient with progressive cytomegalovirus (CMV) disease due to a resistant CMV strain emerging under ganciclovir (GCV) therapy. A discriminative polymerase chain reaction (PCR) assay, designed to detect the resistance-related V460 mutation within the viral enzyme UL97, revealed the presence of a mutated strain in a heterogeneous isolate 51 days after transplantation. The conventional antiviral susceptibility assay had failed to demonstrate resistance to GCV. Under prolonged GCV therapy, the mutated strain dominated the wild-type strain, as shown by the PCR assay. This domination led to laboratory resistance, associated with recurrent fever and progressively severe retinitis. As this discriminative PCR assay was shown to be effective in detecting mutated strains that constitute a minority in the virus load, it should allow better management of patients with CMV disease.