With balanced safety-efficacy profile, letermovir anti-cytomegalovirus (CMV) prophylaxis is used in hematopoietic stem cell transplant recipients (HSCTR). We assessed feasibility and usefulness of letermovir therapeutic drug monitoring (TDM) in HSCTR. We performed a prospective observational study on letermovir-TDM including 40 consecutive adult CMV-seropositive allogeneic-HSCTR who received orally (PO) administered letermovir. Minimal blood concentrations of letermovir (C_trough) were measured on days 3 and 7 postletermovir initiation and weekly thereafter. Letermovir-C_trough remained stable during the first 70 days post-HSCT at a median of 286 μg/L (interquartile range, 131 to 591 μg/L), with large interpatient/intrapatient variability. No associations between breakthrough clinically significant CMV infection or detectable CMV DNAemia and letermovir-C_trough were observed. Patients with letermovir-associated adverse events had higher letermovir-C_trough than patients without (400 versus 266 μg/L, P = 0.02). Letermovir-C_trough was similar in patients with or without gastrointestinal symptoms (280 versus 300 μg/L, P = 0.49). Acute grade ≥2 GvHD was associated with higher letermovir-C_trough (479 versus 248 μg/L, P = 0.001), including gastrointestinal GvHD (499 versus 263 μg/L, P = 0.004). Concomitantly administered posaconazole and cyclosporine were associated with higher letermovir-C_trough (707 versus 259 μg/L, P < 0.001 and 437 versus 248 μg/L, P = 0.01, respectively). In multivariable analysis, both posaconazole (odds ratio [OR], 4.9; 95% confidence interval [CI], 2.4 to 9.7; P < 0.0001) and cyclosporine-adjusted letermovir dose at 240 mg daily (OR, 3.5; 95% CI, 1.4 to 9.0; P = 0.01) were independently associated with higher letermovir-C_trough. In conclusion, administration of PO letermovir led to measurable and relatively stable letermovir-C_trough, without noticeable associations with clinical efficacy. Letermovir exposure was not affected by gastrointestinal symptoms, but with posaconazole and cyclosporine administration. Associations between letermovir and concomitantly administered agents and adverse events warrant additional clinical studies.