We studied the utility and clinical relevance of RUNX1 (runt-related transcription factor 1) mutations and their application as residual disease detection markers using next-generation deep-sequencing. Mutation screening was prospectively performed in 814 acute myeloid leukemia patients. At diagnosis, 211/814 (25.9%) patients harbored mutations with a median clone size of 39%(range: 2–96%). Furthermore, in 57 patients paired samples from diagnosis and relapse were analyzed. In 47/57 (82.5%) cases the same alterations detected at diagnosis were present at relapse, whereas in 1/57 (1.8%) cases the mutation from the diagnostic sample was no longer detectable. Discrepancies were observed in 9/57 (15.8%) cases, also including the occurrence of novel RUNX1 mutations not restricted to those regions affected at diagnosis. Moreover, in 103 patients the prognostic impact of residual levels of RUNX1 mutations during complete remission was studied. Separation of patients according to median residual mutation burden into ‘good responders’ and ‘poor responders’(median: 3.61%; range: 0.03–48.0%) resulted in significant differences of both event-free (median 21.0 vs 5.7 months, P< 0.001) and overall survival (OS; median 56.9 vs 32.0 months, P= 0.002). In conclusion, deep-sequencing revealed that RUNX1 mutations qualify as patient-specific markers for individualized disease monitoring. The measurement of mutation load may refine the assignment into distinct risk categories and treatment strategies.