Efficacy and safety of letermovir as prophylaxis for clinically significant cytomegalovirus infections (csCVMi) was evaluated in randomised controlled trials while most of the real-world studies are single-centre experiences. We performed a retrospective, multi-centre case-control study at six German university hospitals to evaluate clinical experiences in patients receiving CMV prophylaxis with letermovir (n=200) compared to controls without CMV prophylaxis (n=200) during a 48-week follow-up period after allogeneic hematopoietic cell transplantation (aHCT). The incidence of csCMVi after aHCT was significantly reduced in the letermovir (34%, n=68) compared to the control group (56%, n=112; p<0.001). Letermovir as CMV prophylaxis (OR 0.362) was found to be the only independent variable associated with theprevention of csCMVi. Patients receiving letermovir showed significantly better survival compared to the control group (HR = 1.735, 95% CI: 1.111-2.712; p=0.014). Of all csCMVi, 46% (n=31) occurred after discontinuation of letermovir prophylaxis. Severe neutropenia (<500 neutrophils/µL) on the day of the stem cell infusion was the only independent variable for an increased risk of csCMVi after the end of letermovir prophylaxis. Our study highlights the preventive effects of letermovir on csCMVi after aHCT. A substantial proportion of patients developed a csCMVi after discontinuation of letermovir. In particular, patients with severe neutropenia require specific attention after drug discontinuation.