The clinical impact of human cytomegalovirus (CMV) and progression to CMV disease in immunocompromised patients has been reduced by therapeutic strategies using ganciclovir, valganciclovir, foscarnet and cidofovir. However, extensive antiviral therapy increases the risk of antiviral resistance due to mutations in the UL97 protein kinase and UL54 DNA polymerase. Co-infection with HHV-6 or HHV-7 is also associated with increased CMV reactivation and disease. Genotypic CMV antiviral resistance was identified in 38% of Australian immunocompromised patients. While UL97 mutations only were identified in 23% of patients, additional UL54 mutations, with the potential to confer multidrug resistance, were detected in 15% of patients. Antiviral resistant CMV strains were found to emerge rapidly in highly immunocompromised patients, and some strains were able to persist in the absence of selective pressure. Three new mutations were identified (UL97 - N597D, UL54 - F412S, D485N). N597D was characterised by recombinant phenotyping and conferred minimal ganciclovir resistance. Neither baculovirus nor coupled transcription/translation yielded full-length UL54 protein (pUL54; ~140 kDa) for activity assays. However, truncated pUL54 (~66 kDa) was purified after prokaryotic expression. HHV-6 and HHV-7 co-infection was a common clinical occurrence; with 36% of liver transplant recipients infected with HHV-6 (11% persistent) and 80% with HHV-7 (52% persistent). ValGCV therapy did not significantly alter the incidence of HHV-6, HHV-7 or co-infection. The most prevalent co-infection pattern was CMV, HHV-6 and HHV-7 (46%) and both CMV and HHV-7 (38%). CMV reactivation was predominantly independent of HHV-6/HHV-7, although 27% of patients had initial HHV-7 reactivation. Despite frequent co-infection, HHV-6 and HHV-7 were not associated with clinical disease, with possible exception of HHV-7 and acute cellular rejection. CMV antiviral resistance remains a significant issue in transplantation, emphasising the importance of antiviral resistance testing in an era of widespread prophylaxis. New mutations in UL97 and UL54 continue to be identified. Further characterisation of UL54 mutations using polymerase activity assays would increase our knowledge of enzymological basis of antiviral resistance. Co-infection with HHV-6 and HHV-7 is common in transplant recipients, but does not play a significant role in disease. Similar co-infection rates between valGCV-treated and untreated patients indicate that valGCV is not highly effective against HHV-6 and HHV-7.