Background: Primary mediastinal large B-cell lymphoma (PMBCL) is a type of aggressive large B-cell lymphoma which affects mainly young patients and is characterized by a local spread and development of a tumor conglomerate in the mediastinum. One of the most important pathogenetic mechanisms of the development of PMBCLs is their genetic heterogenety. There are a lot of similarities between molecular profiles of diffuse large B-cell lymphoma, Hodgkin’s lymphoma, and gray zone lymphoma, which are described in the literature. However, there is no clear molecular-genetic characteristic of PMBCL. Despite the successful treatment of patients with modern methods of immunochemotherapy, a large number of patients are resistant to the therapy due to the tumor heterogeneity. The aim of this study was the detection of a clinically significant mutation for patients with PMBCL by next-generation sequencing. Aims: To identify significant mutations in the genes in patients with PMBCL for the further studies. Methods: The study included 47 patients diagnosed with different stages of PMBCL who received treatment from 2013 to 2021. NGS was performed using the AVENIO Tumor Expanded Panel, Roche, USA, which includes 77 genes. DNA was isolated using the Gene Read DNA FFPE Kit panel, Qiagen, USA. The data were processed using the AVENIO Oncology Analysis Software to search for clinically significant mutations. The mutations were also confirmed by Sanger sequencing. Results: The genetic material of selected patients was analyzed for the presence of clinically significant mutations among the known databases: COSMIC: v83, TCGA 9.0, ExAC:1.0, dbSNP:150, 1000 Genomes: phase_3_v5b, SnpEff:4.2. A missense variant of clinically significant mutations in the FGFR3, PIK3CA, ALK, EZH2, RNF43, MET genes was identified in patients with refractory PMBCL. For example, an activating driver mutation in the FGFR3 gene was detected in the 9th exon (rs121913479), which is pathogenic. The frequency of alleles was 25.54%. An activating driver mutation in the PIK3CA gene was detected in the 10th exon (rs121913273), which is also pathogenic. The frequency of alleles was 10.88%. The aim of the study was to identify signaling pathways which were related to mutated genes. They were PI3K-AKT, JAK-STAT, RAP1, RAS, MAPK, stem cell regulation signaling pathway, which were also interpreted. According to the bioinformatics analysis, FGF/FGFR3 was the most significant signaling pathway for PMBCL in our study, because it is able to activate all listed pathways via FGFR-receptor activation. Summary/Conclusion: NGS revealed clinically significant mutations in the genes in patients with PMBCL, the therapeutic potential of which requires the further studies.