Molecular routine diagnostics for BCR-ABL1-negative myeloproliferative neoplasms (MPN) currently focusses on mutations in JAK2, CALR and MPL. In recent years, recurrent mutations in MPNs have been identified in several other genes. We here present the validation of a next generation sequencing (NGS)-based 28-gene panel and its use in MPN. We analysed the mutation status of 28 genes in 100 MPN patients [40 essential thrombocythaemia (ET), 30 primary myelofibrosis (PMF), 30 polycythaemia vera (PV)] and found two or more mutated genes in 53 patients. Moreover, significantly more mutated splicing genes (SF3B1, SRSF2 and U2AF1) were present in PMF (060 mutated genes/patient) compared to ET (015) while no mutations in splicing genes were found in PV. Additionally, chromatin modification genes (ASXL1 and EZH2) were frequently mutated in PMF patients (050) and, to a significantly lesser extent, in ET (013) and PV (007). Contrarily, DNA methylation genes (DNMT3A, IDH1, IDH2 and TET2) were mutated most often in PV (05) and less frequently in ET (023) and PMF (020), but without reaching statistical significance. Our results demonstrate the feasibility and utility of NGS-based panel diagnostics for MPN. With 53% of the patients bearing two or more mutated genes, their prognostic relevance needs further studies.