Introduction <p><i>FMS</i>-like tyrosine kinase 3 (<i>FLT3</i>) mutations show variable detectability in relapse/refractory acute myeloid leukemia (AML) with unclear clonal evolution dynamics.</p> Methods <p>This prospective noninterventional study examined clonal evolution and outcomes from AML diagnosis to relapse/refractory disease occurrences.</p> Results <p>Of 650 patients included, 172 were <i>FLT3</i>-positive (<i>FLT3</i>^pos) and 472 were <i>FLT3</i>-negative (<i>FLT3</i>^neg; 99.1% testing rate; unknown <i>FLT3</i> status, six patients). At first occurrence, the <i>FLT3</i> testing rate decreased (57.0% [166/291]). Among tested patients, 45 had <i>FLT3</i>^pos and 121 had <i>FLT3</i>^neg AML. A gain or loss of mutations was seen in 15.6% (7/45) of patients with <i>FLT3</i>^pos AML and 14.9% (18/121) of patients with <i>FLT3</i>^neg AML. Median (95% confidence interval [CI]) overall survival was 22.8 (19.6, not estimable [NE]) months across patients (<i>FLT3</i>^pos, NE;<i>FLT3</i>^neg, 20.3 [15.2–23.7] months; hazard ratio [HR] [95% CI] 0.6 [0.5–0.8]). Median (95% CI) disease-free survival across patients was NE (26.3–NE) (<i>FLT3</i>^pos, NE;<i>FLT3</i>^neg, NE; HR [95% CI] 0.8 [0.6–1.1]). Median event-free survival (95% CI) was 11.8 (10.0–15.5) months in all patients (<i>FLT3</i>^pos, 17.2 [11.0–NE] months;<i>FLT3</i>^neg, 10.4 [8.4–13.2] months; HR [95% CI] 0.8 [0.6–1.0]).</p> Conclusions <p>Dynamic changes in <i>FLT3</i> mutation status were observed during these patients’ disease course. <i>FLT3</i>^pos status at baseline, but not at first occurrence, was associated with improved outcomes; other confounders should be considered. Timelier <i>FLT3</i> mutation retesting may aid in personalizing treatment.&#xa0;Graphical abstract available for this article.</p> Graphical Abstract <p></p>