Objective Chimerism is well-established for surveillance of acute myeloid leukemia (AML) patients after allogeneic hematopoietic stem cell transplantation (HSCT), but interpretation of the results and techniques is not standardized. Materials and Methods We correlated chimerism in 75 AML patients (38 male, 37 female) who underwent myeloablative (n = 36)/reduced (n = 39) allo-HSCT with the risk of relapse and survival. Chimerism was evaluated by quantitative real-time polymerase chain reaction for donor/recipient specific polymorphisms/Y-specific sequences. Results After HSCT, 40 patients (53%) achieved stable complete donor chimerism (≥99.0% of donor alleles), while 35 (47%) failed to achieve stable donor chimerism. Thirty-one patients (41%) showed decreasing donor alleles after having first achieved complete donor chimerism. To investigate the kinetics of mixed chimerism, patients were separated whether they showed subsequent increasing or decreasing donor alleles. Subsequent decrease of donor alleles was associated with relapses in 17 of 18 cases (94%), while no patient with subsequent increasing donor alleles relapsed ( p < 0.001). Patients with mixed chimerism and increasing donor alleles had better 2-year disease-free survival (85%) than those with decreasing donor alleles (0%; p < 0.001). Conclusions The kinetics of mixed chimerism as assessed by quantitative real-time polymerase chain reaction is an important prognostic predictor in the post-transplantation period of AML patients.