As the impact of targeted next-generation sequencing (TNGS) on daily diagnosis has not been evaluated, we performed TNGS (46 genes) on lymphomas of unclear subtype following expert haematopathological review. The potential impact on patient care and modifications of final diagnosis were divided into major and minor changes according to the European Society of Medical Oncology (ESMO) guidelines. Among 229 patients [19 primary central nervous system lymphomas (PCNSL), 48 large B-cell lymphomas (LBCLs), 89 small BCLs (SBCLs), seven Hodgkin lymphomas (HL), 66 T-cell lymphomas], the overall concordance rate of histological and TNGS diagnosis was 89Á5%. TNGS confirmed the histological diagnosis in 144 cases (62Á9%), changed the diagnosis in 24 cases (10Á5%) and did not help to clarify diagnosis in 61 cases (26Á7%). Modifications to the final diagnosis had a clinical impact on patient care in 8Á3% of cases. Diagnostic modifications occurred in all types of lymphoma except in PCNSL and HL; the modification rate was 14Á6% in SBCL and 12Á5% in LBCL. While comparing informative and uninformative cases, no differences were found in terms of DNA amplification, quality or depth of sequencing and biopsy type. The present study highlights that TNGS may directly contribute to a more accurate diagnosis in difficult-to-diagnose lymphomas, thus improving the clinical management in routine practice.