This study evaluated azacitidine as treatment of minimal residual disease (MRD) determined by a sensitive donor chimerism analysis of CD34⁺ blood cells to pre-empt relapse in patients with CD34⁺ myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT). At a median of 169 days after HSCT, 20/59 prospectively screened patients experienced a decrease of CD34 ⁺ donor chimerism to <80% and received four azacitidine cycles (75 mg/m²/day for 7 days) while in complete hematologic remission. A total of 16 patients (80%) responded with either increasing CD34⁺ donor chimerism to ≥80% (n=10; 50%) or stabilization (n=6; 30%) in the absence of relapse. Stabilized patients and those with a later drop of CD34 ⁺ donor chimerism to <80% after initial response were eligible for subsequent azacitidine cycles. A total of 11 patients (55%) received a median of 4 (range, 1-11) additional cycles. Eventually, hematologic relapse occurred in 13 patients (65%), but was delayed until a median of 231 days (range, 56-558) after initial decrease of CD34⁺ donor chimerism to <80%. In conclusion, pre-emptive azacitidine treatment has an acceptable safety profile and can substantially prevent or delay hematologic relapse in patients with MDS or AML and MRD after allogeneic HSCT.