Differential diagnosis of chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML) and chronic neutrophilic leukemia (CNL) is not always straightforward as they share several features. Over the last years, a number of molecular markers have been identified that are frequently mutated in these myeloid neoplasms. Mutations of SETBP1 in aCML and ASXL1 in CMML are described to confer an adverse prognosis. We retrospectively analyzed 123 patients with myeloid malignancies for hotspot mutations in SETBP1 (p.Leu790-p.Glu965), ASXL1 (p.Ile574-p.Leu743) and CSF3R (p.Ala576-p.Pro621;p.Asp681-p.Glu808) using Sanger sequencing. Of these, 43 patients were diagnosed with MDS, 49 with MDS/MPN and 31 with MPN. In the MDS/MPN cohort, 13 patients were diagnosed with aCML and 28 with CMML. Only one case of CNL was included. Mutation frequencies of SETBP1 in aCML and CMML are in accordance with literature. SETBP1 mutations closely relate to aCML. We confirm the findings of other reports that ASXL1 mutations are frequent in all myeloid malignancies. Mutation frequency of ASXL1 in aCML was particularly high (53.8%). The observation of Maxson et al. [1] of the presence of CSF3R mutations in 40% of patients with aCML cannot be confirmed, but our study is in agreement with two other reports [2,3]. No conclusions can be made on mutation frequencies of CSF3R in CNL. This study has shown that hotspot mutation analysis of SETBP1, ASXL1 and CSF3R can be helpful to guide diagnosis of myeloid malignancies.