Primary central nervous system lymphoma (PCNSL) is a rare and special type of non-Hodgkin lymphoma with a significantly worse median overall prognosis than that of non-Hodgkin lymphoma outside the brain. Clarifying the genomic characteristics and alterations in PCNSL could provide clues regarding its distinctive pathophysiology and new treatment options. However, current knowledge about the genomics of PCNSL is limited. In this study, next-generation sequencing (NGS) was performed to investigate the genomic profile of PCNSL. Samples from 12 patients diagnosed with PCNSL at our institution were analyzed for gene mutations using NGS. This study showed that missense mutations were the most common mutation type. C > A/G > T accounted for most of the single-base mutations, which reflected the preference of the tumor sample mutation type and may serve as an important prognostic factor. The most significantly mutated gene was myeloid differentiation factor 88 (MYD88) (0.55), followed by CD79B, LRP1B, and PRDM1 (0.36). None of the cases showed a high tumor mutational burden. In addition to the traditional driver genes, we also identified some new possible ones such as MET, PIM1, and RSBN1L. Enrichment analysis revealed that genes mutated in PCNSL were involved in many pathways and functional protein activities, such as the extracellular matrix and adhesion molecules. The most common genetic alterations in PCNSL were identified using NGS. Mutations in multiple genes highlights the complex molecular heterogeneity of PCNSL. Enrichment analysis revealed possible pathogenesis. Further exploration of new driver genes could provide novel insights into diagnosis and precision medicine for PCNSL.Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.