Background: Gene mutations play key role in the transformation from myelodysplastic syndrome (MDS) to secondary acute myeloid leukemia (s-AML). The aim of this study was to explore the clonal progression pattern from MDS to s-AML. Methods: A total of 663 patients diagnosed with MDS (n = 291), s-AML (n = 86), and primary AML (p-AML) (n = 286, M3 excluded) were enrolled in this study from May 2018 to December 2021, and the median age was 57 year-old (range 1~88). The diagnosis was established according to the WHO criteria. The MDS patients were divided into low-risk MDS ( n = 158) and high-risk MDS (n = 133) according to 2012 IPSS-R. Next generation sequencing was performed to detect the mutations of entire coding regions of 112 genes related with hematologic tumors in all patients. Results: It was found that mutations from 31 genes occurred in both MDS and AML, including DNA methylation (DNMT3A, TET2, IDH1, IDH2, WT1), chromatin modification (SETBP1,ASXL1, EZH2, PHF6), RNA splicing (U2AF1, SF3B1, SRSF2, ZRSR2), transcription regulation (TP53, CEBPA,RUNX1, ETV6, GATA2, BCOR, BCORL1), signal transduction pathways (NRAS, KRAS, PTPN11, NOTCH1, FLT3, JAK2, KIT), and others (NPM1, FAT, STAG2, DDX41). By enrichment analysis, those mutations were classified into two categories: type A mutations (FLT3, IDH1, NPM1, NRAS, PTPN11, WT1, CEBPA) and type B mutations (IDH2, KRAS, U2AF1, ZRSR2, SRSF2, STAG2, DNMT3A, EZH2, RUNX1, ETV6, TP53). The type A genes were significantly enriched in p-AML than in MDS, while some type B mutations, particularly RNA splicing, were significantly enriched in MDS than in p-AML. The VAFs of type A mutations were significantly lower than those of type B mutations. The longitudinal analysis of 10 patients with s-AML revealed two main models of clonal evolution, linear evolution and clone sweeping. Although the two models differed in the disease development, new sub-clones appeared in 9/10 patients. Interestingly, many of the new mutations were type A mutations (n = 7, 7/10), and 7 out of 10 patients carried type B mutations when they were at MDS state. The VAF data also showed that the preexisting mutations were generally higher than the newly emerging genetic alterations. Finally, an individual case was used to delineate the clone evolution. The patient was initially diagnosed with 5q syndrome and U2AF1 mutation (type B) was identified. After treatment, the patient experienced remission, and developed a new ETV6 mutation while the clonal size of U2AF1 decreased. When the patient relapsed, the clone size of ETV6 decreased while U2AF1 mutated clone size were amplified, a typical “clone sweeping” pattern. When the patient eventually transformed to s-AML, three type A mutations (NRAS, KRAS and PTPN11) appeared simultaneously. Conclusions: This study revealed that the type A mutations are mainly responsible for the progression of MDS to AML, and the type B mutations are related to the clonal origin of MDS.