AbstractTo refine the biological and prognostic significance ofDNMT3Amutations in acute myeloid leukemia (AML), we assessed the impact ofDNMT3Avariant allele frequency (VAF) and its comutations in this study. Using targeted next‐generation sequencing, we analyzed 171 adult patients with de novo cytogenetically normal AML forDNMT3Amutations and associated comutations.DNMT3Amutwas detected in 35 patients.DNMT3Amutpatients were divided intoDNMT3AHighandDNMT3ALowusing a cut‐off VAF value of 42%. We observed thatDNMT3AHighpatients at diagnosis had increasing white blood cell (WBC) counts (p< 0.001) and a higher lactate dehydrogenase (LDH) level (p= 0.027), and were associated with lower complete remission (CR) rate (p= 0.015) and shorter overall survival (OS) (p= 0.032) thanDNMT3ALowpatients. We classified two different comutated genetypes, includingDNMT3AmutNPM1mutFLT3‐ITDmutandDNMT3AmutIDH1/IDH2mut. Patients withDNMT3AmutNPM1mutFLT3‐ITDmutshowed worse OS (p= 0.026) and relapse‐free survival (RFS) (p= 0.003) than those withDNMT3AmutIDH1/IDH2mut, and showed a shorter OS (p= 0.027) than those withDNMT3AwtNPM1mutFLT3‐ITDmut. We also observed that patients withDNMT3AmutIDH1/IDH2muthad higher platelet counts (p= 0.009) and a lower BM blast percentage (p= 0.040) than those withDNMT3AwtIDH1/IDH2mut. In multivariate analyses,DNMT3AHighwas independently associated with a lower CR rate (OR = 5.883;p= 0.004) and shorter OS (HR = 3.768;p <0.001).DNMT3AmutNPM1mutFLT3‐ITDmutindependently affected worse OS (HR = 6.030;p <0.001) and RFS (HR = 8.939;p <0.001). Our findings might be potentially useful for predicting clinical outcomes.