Abstract: The objective of this study was to investigate which comutations based on next-generation sequencing (NGS) at diagnosis affect the clinical prognosis of de novo AML patients with FLT3-ITD mutations and the effect of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) on the comutations. We analyzed 95 de novo AML patients with FLT3-ITD mutations from January 2018 to August 2021 based on the NGS 99-gene platform. Forty-one other types of molecular mutations were detected. The most common cooccurring mutations were NPM1 (n = 43, 45.3%) and DNMT3A (n = 21, 22.1%). NPM1 mutation status did not affect the clinical outcomes. AML patients with FLT3-ITD and DNMT3A comutations had significantly worse 3-year DFS (25.2% and 62.6%, P = 0.003) and OS rates (57.3% vs. 73.1%, P = 0.047) than those without DNMT3A mutations and the survival was significantly more favorable after haplo-HSCT than chemotherapy (3-year DFS,77.1% vs. 15.4%, P = 0.009; 3-year OS, 82.8% vs. 46%, P = 0.001, respectively). By multivariate analysis, DNMT3A mutation was a risk factor for DFS and OS, while haplo-HSCT was a protective factor. DNMT3A mutation might be a poor prognostic factor in adult AML patients with FLT3-ITD mutations and haplo-HSCT could overcome the poor prognostic of DNMT3A comutation.