Abstract Objectives Antiviral resistance in cytomegalovirus (CMV) may result from mutations in the molecular targets of antiviral agents. The aim of this study was to investigate both the prevalence of resistance-associated mutations and the factors associated with antiviral resistance in solid organ transplant (SOT) patients with repeated high CMV loads during antiviral treatment. Methods SOT patients were selected retrospectively, based on CMV loads of >30000 IU/mL at least twice in a period during which treatment was given. Patient samples were tested for antiviral resistance by Sanger sequencing the UL97 and UL54 genes of CMV, which code for the viral kinase and polymerase. Factors predisposing to and resulting from the development of antiviral resistance mutations were analysed. Results Multiple samples from 113 SOT patients were tested, showing resistance-associated mutations in 25 patients (22%). A further 20 (18%) patients showed mutations that were not known to be associated with antiviral resistance. Several factors were associated with development of resistance-associated mutations in UL97 as well as UL54, including human leucocyte antigen (HLA) mismatch, which occurred more frequently in the group of patients with resistance mutations. High-level resistance mutations were most frequently seen in UL97. Conclusions This study shows that by selecting patients solely on the basis of virological response to treatment, more patients with antiviral resistance mutations are identified. In this study we confirm findings by other groups that primary infections are associated with resistance development. Moreover, we show that HLA mismatch is associated with the development of antiviral resistance, which suggests a role for host immunity in the development of resistance.