AbstractAlthough most patients with acute myeloid leukemia (AML) achieve clinical remission with induction chemotherapy, relapse rates remain high. Next-generation sequencing enables minimal/measurable residual disease (MRD) detection; however, clinical significance is limited due to difficulty differentiating between pre-leukemic clonal hematopoiesis and frankly malignant clones. Here, we investigated AML MRD using targeted single-cell sequencing (SCS) at diagnosis, remission, and relapse (n = 10 relapsed, n = 4 nonrelapsed), with a total of 310 737 single cells sequenced. Sequence variants were identified in 80% and 75% of remission samples for patients with and without relapse, respectively. Pre-leukemic clonal hematopoiesis clones were detected in both cohorts, and clones with multiple cooccurring mutations were observed in 50% and 0% of samples. Similar clonal richness was observed at diagnosis in both cohorts; however, decreasing clonal diversity at remission was significantly associated with longer relapse-free survival. These results show the power of SCS in investigating AML MRD and clonal evolution.