Acute myeloid leukemia (AML) has been thought to be the consequence of two broad complementation classes of mutations: class I and class II. However, overlap-mutations between them or within the same class and the position of TP53 mutation are not fully analyzed. We comprehensively analyzed the FLT3, cKIT, N-RAS, C/EBPA, AML1, MLL, NPM1, and TP53 mutations in 144 newly diagnosed de novo AML. We found 103 of 165 identified mutations were overlapped with other mutations, and most overlap-mutations consisted of class I and class II mutations. Although overlap-mutations within the same class were found in seven patients, five of them additionally had the other class mutation. These results suggest that most overlap-mutations within the same class might be the consequence of acquiring an additional mutation after the completion both of class I and class II mutations. However, mutated genes overlapped with the same class were limited in N-RAS, TP53, MLL-PTD, and NPM1, suggesting the possibility that these irregular overlap-mutations might cooperatively participate in the development of AML. Notably, TP53 mutation was overlapped with both class I and class II mutations, and associated with morphologic multilineage dysplasia and complex karyotype. The genotype consisting of complex karyotype and TP53 mutation was an unfavorable prognostic factor in entire AML patients, indicating this genotype generates a disease entity in de novo AML. These results collectively suggest that TP53 mutation might be a functionally distinguishable class of mutation.