Abstract Cytomegalovirus (CMV)-seropositive umbilical cord blood transplantation (CBT) recipients have a high incidence of CMV-associated complications. There are limited data regarding the efficacy of letermovir for preventing clinically significant CMV infection (CS-CMVi), and the impact of letermovir prophylaxis on delayed-onset CMV reactivation after letermovir discontinuation, in CBT recipients. We compared the cumulative incidence of CS-CMVi and CMV detection in 21 CMV-seropositive CBT recipients receiving letermovir prophylaxis with a historical cohort of 40 CBT recipients receiving high-dose valacyclovir prophylaxis. Letermovir was administered on day +1 up to day +98. The cumulative incidence of CS-CMVi was significantly lower by day 98 in the letermovir cohort (19% vs 65%). This difference was lost by 1 year due to a higher incidence of delayed-onset CMV reactivation in the letermovir cohort. No patients developed CMV disease in the letermovir cohort within the first 98 days compared with 2 cases (2.4%) in the high-dose valacyclovir cohort; 2 patients developed CMV enteritis after discontinuing letermovir. Median viral loads were similar in both cohorts. Thus, letermovir is effective at preventing CS-CMVi after CBT, but frequent delayed-onset infections after letermovir discontinuation mandate close monitoring and consideration for extended prophylaxis.