Relapse is a major complication of acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (SCT). The objective of our study was to evaluate chimerism monitoring on the CD3-negative mononuclear cells by RQ-PCR to predict relapse of patients allografted for AML and to compare its performance with WT1 quantification. A cohort of 100 patients undergoing allogenic SCT for AML was retrospectively analyzed in a single institution. Patients without complete chimerism, defined as less than 0.01% of recipient's DNA in CD3-negative cells, had a significantly higher risk of relapse and a lower overall survival (p < 0.001). An increase in the percentage of recipient DNA in CD3-negative cells was associated with an increased risk of relapse (p < 0.001) but not with overall survival. Comparable performances between monitoring of CD3-negative cell chimerism and WT1 expression to predict relapse was observed up to more than 90 days before hematological relapse, with sensitivity of 82% and 78%, respectively, and specificity of 100% for both approaches. Quantitative specific chimerism of the CD3-negative mononuclear fraction, enriched in blastic cells, is a new and powerful tool for monitoring measurable residual disease and could be used for AML patients without available molecular markers.