We studied the characteristics of the provisional category de novo acute myeloid leukemia (AML) with mutated RUNX1 (AML-RUNX1) proposed by the World Health Organization (WHO). Until now, most published studies have combined de novo and secondary AML-RUNX1. We compared the clinicopathologic characteristics and outcomes of WHO-defined de novo AML-RUNX1 with de novo AML without RUNX1 alterations (AML-RUNX1). We performed sequential NGS to assess RUNX1 mutation stability over disease course. We identified 46 de novo AML-RUNX1 patients [32 (70%) men, 14 (30%) women; median age, 66.5 years] with 54 RUNX1 mutations [median VAF, 32% (2-97%)]. Point mutations clustered within the runt-homology-domain and frame-shift mutations within the transactivation domain. Compared with AML-RUNX1, AML-RUNX1 showed male predominance (p = 0.02), higher frequency of SRSF2 (p = 0.02), and ASXL1 (p = 0.0004) mutations and normal karyotype (p = 0.01), and absent NPM1 mutations (p = 0.0002). De novo AML-RUNX1 showed no significant difference in overall survival (OS) compared with AML-RUNX1 (median: 26 vs. 32 months) (p = 0.71). AML-RUNX1 with clonal RUNX1 mutation (≥20% VAF) had shorter OS than subclonal <20% VAF (23 months vs. undefined; p = 0.04). However, the difference was not significant when compared with AML-RUNX1 (23 vs. 32 months; p = 0.23). No significant OS difference was noted between de novo AML-RUNX1 and AML-NOS-RUNX1. By sequential multigene mutation profiling, RUNX1 mutation disappeared at relapse in one of ten patients. Overall, the findings support separate categorization of this entity. However, there is no significant outcome difference compared with AML-RUNX1.