Chromosomal translocations resulting in fusion genes represent important oncogenic drivers and potential therapeutic targets in rare leukemia subtypes. Formalin-fixed and paraffin-embedded trephines are frequently used in hematologic diagnostic and provide relevant access to leukemic cells for further studies, e.g. phenotyping in bone marrow fibrosis. However, high-throughput molecular analysis of nucleic acids obtained from this material is challenging, especially the reliable detection of RNA transcripts. We analyzed 63 formalin-fixed and paraffin-embedded bone marrow trephines of patients with chronic eosinophilic leukemia, chronic myeloid leukemia, acute myeloid leukemia and myeloproliferative neoplasms for gene mutations and the presence of fusion transcripts with a commercial amplicon-based next generation sequencing approach. Thereby we could detect and validate (by reverse transcription PCR) fusion transcripts relevant for diagnosis and therapy in 25 patients (39.7%). Retrospectively selected material, up to 10 years old, was used for this purpose and only one sample failed in the RNA analysis (1.6%). We conclude that amplicon-based fusion transcript detection in bone marrow trephines is feasible and that bone marrow trephines taken for histologic assessment can also be applied for high-throughput molecular analysis.Copyright © 2020. Published by Elsevier Inc.