We reported a ganciclovir (GCV)-resistant cytomegalovirus (CMV) infection in a heart transplant recipient. Genotypic and phenotypic susceptibility assays demonstrated an A594V mutation in the UL97 phosphotransferase gene and GCV IC(50)>96 microM. Low GCV concentration exposure, immunosuppressive treatment, donor-positive/recipient-negative CMV serostatus, viral reactivations within antiviral prophylaxis or treatment, contributed to GCV-resistant strain selection.