The mutational spectrum and molecular characteristics of acute myelomonocytic lineage leukemia, namely acute myeloid leukemia (AML) French-American-British (FAB) subtypes M4 and M5, are largely unknown. In order to explore the mutational spectrum and prognostic factors of FAB-M4 and -M5, next-generation sequencing (NGS) was performed to screen for mutated genes and fusion genes relevant to the pathogenesis of AML. Of the 63 patients enrolled in the study, 60% had more than three mutated genes. NPM1 had the highest mutation frequency, followed by DNMT3A, FLT3, NRAS, RUNX1, and TET2. Univariate analysis suggested that age ≥60 years was an independent factor for both poor event-free survival (EFS) and overall survival (OS, P = 0.009, 0.002, respectively), MYH11-CBFβ was associated with better EFS and OS (P = 0.029, 0.016, respectively). However, multivariate analysis was not able to identify any independent risk factor for survival in the cohort of FAB-M4 and -M5 patients, including peripheral white blood cell count, bone marrow blast percentage, MYH11-CBFβ, FLT3-ITD, mutations in NPM1 and DNMT3A, and allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our study provided new insight into the mutational spectrum and molecular characteristics of FAB-M4 and -M5. The clinical implications of the genetic signature of FAB-M4 and -M5 need to be further elucidated by larger studies.