The study of human cytomegalovirus (HCMV) antiviral drug resistance has enhanced knowledge of the virological targets and the mechanisms of antiviral activity. The currently approved drugs, ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV), target the viral DNA polymerase. GCV anabolism also requires phosphorylation by the virus-encoded UL97 kinase. GCV resistance mutations have been identified in both genes, while FOS and CDV mutations occur only in the DNA polymerase gene. Confirmation of resistance mutations requires phenotypic analysis; however, phenotypic assays are too time-consuming for diagnostic purposes. Genotypic assays based on sequencing provide more rapid results but are dependent on prior validation by phenotypic methods. Reports from many laboratories have produced an evolving list of confirmed resistance mutations, although differences in interpretation have led to some confusion. Recombinant phenotyping methods performed in a few research laboratories have resolved some of the conflicting results. Treatment options for drug-resistant HCMV infections are complex and have not been subjected to controlled clinical trials, although consensus guidelines have been proposed. This review summarizes the virological and clinical data pertaining to HCMV antiviral drug resistance.