Single-cell DNA sequencing can address the sequence of somatic genetic events during myeloid transformation in relapsed acute myeloid leukemia (AML). We present an NPM1-mutated AML patient with an initial low ratio of FLT3-ITD (low-risk ELN-2017), treated with midostaurin combined with standard chemotherapy as front-line treatment, and with salvage therapy plus gilteritinib following allogenic stem cell transplantation after relapse. Simultaneous single-cell DNA sequencing and cell-surface immunophenotyping was used in diagnostic and relapse samples to understand the clinical scenario of this patient and to reconstruct the clonal composition of both tumors. Four independent clones were present before treatment: DNMT3A/DNMT3A/NPM1 (63.9%), DNMT3A/DNMT3A (13.9%), DNMT3A/DNMT3A/NPM1/FLT3 (13.8%), as well as a wild-type clone (8.3%), but only the minor clone with FLT3-ITD survived and expanded after therapy, being the most represented one (58.6%) at relapse. FLT3-ITD was subclonal and was found only in the myeloid blast population (CD38/CD117/CD123). Our study shows the usefulness of this approach to reveal the clonal architecture of the leukemia and the identification of small subclones at diagnosis and relapse that may explain how the neoplastic cells can escape from the activity of different treatments in a stepwise process that impedes the disease cure despite different stages of complete remission.