•Rarely reported KMT2D, FAT1, and RELN mutations were detected at high frequencies in Chinese AML patients for the first time.•The gene mutation spectrum of older patients was markedly different to that of younger patients.•Older patients harbored more SRSF2 mutations, which predicted lower rates of overall and relapse-free survival. Differences in the gene mutation spectra of younger and older Chinese adult AML patients and the prognostic significance of these differentially presented gene mutations are rarely reported. One hundred and thirteen newly diagnosed Chinese adults with AML, divided into groups of younger and older patients, were enrolled in this study. Bone marrow samples from the patients were analyzed using targeted next-generation sequencing with a panel of 141 genes. Ninety-eight mutated genes were detected and the top 10 mutated genes were KMT2D, FLT3, FAT1, ASXL1, NRAS, DNMT3A, RELN, TET2, JAK2, and KRAS. The top five functional groups were the tyrosine kinase pathway, transcription factors, DNA methylation, chromatin modifiers, and the JAK-STAT signaling pathway. Younger patients exhibited higher incidences of KMT2D (33.8% vs 10.4%, P = 0.004) and KRAS (15.4% vs 2.1%, P = 0.042) mutations than older patients; whereas, older patients harbored more SRSF2 (20.8% vs 0%, P = 0.002), transcription factor (85.4% vs 67.7%, P = 0.031), DNA methylation (58.3% vs 36.9%, P = 0.024), and RNA splicing (31.3% vs 12.3%, P = 0.013) mutations than younger patients. Moreover, patients with SRSF2 mutations exhibited a lower rate of overall survival (P < 0.001) and relapse-free survival (P < 0.001) than patients carrying wild-type SRSF2. In conclusion, rarely reported KMT2D, FAT1, and RELN mutations were detected at high frequencies in our cohort. The gene mutation spectrum of older patients was different to that of younger patients. Moreover, older patients harbored more SRSF2 mutations, which predicted lower rates of overall and relapse-free survival.