Specific mutations in the human cytomegalovirus (HCMV) DNA polymerase (pUL54) are known to confer resistance against all currently licensed drugs for treatment of HCMV infection and disease. Following the widespread use of antivirals, the occurrence of HCMV drug resistance is constantly increasing. Recently, diagnostic laboratories have started to replace phenotypic drug resistance testing with genotypic resistance testing. However, the reliability and success of genotypic testing highly depend on the availability of high-quality phenotypic resistance data for each individual mutation and for combinations of mutations, with the latter being increasingly found in patients' HCMV isolates. We performed clonal marker transfer experiments to investigate the impacts of 7 different UL54 point mutations and also of combinations of these mutations on drug susceptibility and viral replicative fitness. We show that several mutations-S695T, A972V, K415R, S291P, and A692V-of suspected but uncertain drug susceptibility phenotype, either alone or in combination, were not relevant to antiviral drug resistance. In contrast, the combination of two mutations individually characterized previously-E756K and D413E-conferred high-grade loss of susceptibility to all three antivirals. Our results have been added to the newly available database of all published HCMV resistance mutations (http://www.informatik.uni-ulm.de/ni/mitarbeiter/HKestler/hcmv/index.html). These data will allow better interpretation of genotypic data and further improve the basis for drug resistance testing.