Objective: To characterize the gene mutation in myeloid malignancies with normal karyotype by exploring multiple gene mutations. Methods: We performed targeted second generation sequencing using a custom-designed 49-gene panel, CALR, FLT3 internal tandem duplication (FLT3-ITD), NPM1 and CEBPA mutation were detected by Sanger sequencing. Results: ①Together, gene mutations accounted for a considerable frequency of 98.8% in acute myelogenous leukemia (AML) patients. Coexistence of ≥ 3 mutations was identified in 52.4% patients. The most commonly mutated gene was NPM1 (35.4%), followed by FLT3-ITD (25.6%), CEBPA double mutations (24.4%), DNMT3A (19.5%), TET2(18.3%), NRAS (13.4%), RUNX1(11.0%) and CSF3R (11.0%) mutations. ②The gene mutations were present in 90% myelodysplastic syndrome (MDS) patients. Coexistence of ≥ 3 mutations was in 55.0% patients. The most commonly mutated gene was RUNX1(35.4%), followed by ASXL1(25.0%), SF3B1(15.0%), FLT3-TKD (15.0%) and BCOR (15.0%) mutations. ③The incidence of mutation was similar in AML patients and MDS patients(P=0.097). The mutation rate of each of NPM1 and CEBPA double mutations was higher in patients with AML(P<0.05), while RUNX1, ASXL, SF3B1 and BCOR mutations were identified more frequently in MDS patients, both differences were significant(P<0.05). ④Gene aberrations involved in DNA methylation (DNMT3A, TET2, IDH1/2) and Receptors/kinases (FLT3-ITD，FLT3-TKD，JAK1，JAK2，JAK3，c-KIT，PDGFRA，PDGFRB，MPL，CSF3R，NOTCH1，IL7R) significantly predominated in AML while post-translational chromatin modification (EZH2,ASXL1/2,SETD2) and RNA splicing (SRSF2,SF3B1,ZRSR2,U2AF1) significantly predominated in MDS(P<0.05).