Clinical resistance of cytomegalovirus (CMV) against the currently licensed antiviral drugs is becoming an increasingly recognized problem. This review focuses on the molecular basis of resistance and describes mutations in the UL54 DNA polymerase leading to resistance against cidofovir, foscarnet and ganciclovir. The review highlights two important developments in our appreciation of resistance. Firstly, the use of more rapid molecular based assays to detect genotypic resistance and secondly, the relationship between resistance profiles in multiple organ systems of the same host. Finally, the changing face of CMV disease in the era of highly active antiviral chemotherapy is considered with respect to its impact on the frequency of CMV resistance in the clinic.