Cytomegalovirus (CMV) breakthrough despite therapy with ganciclovir remains a problem, particularly for solid organ transplant (SOT) patients. This could be owing to inadequate ganciclovir blood concentrations, and it is unclear whether monitoring of this is of clinical use. The first study aim was to verify the validity of ganciclovir blood concentration monitoring with daily area-under-curve (AUC24) as a predictive marker for breakthrough CMV. Systematic review of 610 SOT recipients across 11 studies evaluated the association between ganciclovir AUC24 and CMV incidence. Despite dose adjustments for creatinine clearance (CrCL), AUC24 for patients varied from 28-53.7 µg· h/mL. The incidence of CMV infection ranged from 0-50%, while disease ranged from 0-3.1%. One study showed a reduced risk of viraemia when AUC24 was 40-50 µg· h/mL. This formed the basis for a prospective 2-year study on 50 kidney transplant recipients monitored for ganciclovir AUC24 and CMV infection and genotypic resistance markers. Doses given to patients with CrCL< 40 mL/min were higher than the guidelines, and lower when CrCL≥ 40 mL/min. Mean AUC24 was 33±13 µg· h/mL and 82% of subjects did not attain therapeutic target. Monte Carlo simulations with data from the study cohort were conducted to determine whether the manufacturer’s dosing would be predicted to result in higher target attainment than the study cohort. Simulations showed guidelines also could not attain therapeutic target in 80% of individuals. Breakthrough CMV occurred in 6% of recipients, while 12% developed late-onset infection. Mean AUC24 between recipients with and without infection was not significantly different (p= 0.528). One recipient with AUC24 20 µg· h/mL acquired GCV-resistant mutations. The second aim was to uncover novel GCV-resistance mutations. Two UL97 mutations of unknown antiviral susceptibility, A594S and G598D, were selected from the diagnostics laboratory for examination. Investigations found A5946S-mutated CMV showed GCV-resistance on plaque reduction, while virus replication was similar to wildtype. Enzyme activity was assessed using ganciclovir as substrate, or auto-phosphorylation, showed ectopically-expressed UL97 mutants retained some enzyme activity in vitro. In conclusion, ganciclovir AUC24 predicted CMV breakthrough of resistant variants in kidney transplant recipients. CMV genotypes of unknown antiviral susceptibility were evaluated, where marker-transfer provided evidence for a new GCV-resistant CMV UL97 mutation.