Background: Gastrointestinal stromal tumors (GISTs) form the most common mesenchymal tumor of the gastrointestinal tract. The majority of GISTs are characterized by activating mutations in the KIT and PDGFRA genes (80 e 85 %). Approximately 10 e 15 % of adult (and 85 % pediatric) GISTs do not have detectable mutations in KIT or PDGFRA genes (KIT/PDGFRA non-mutated GISTs). KIT/PDGFRA non-mutated GISTs are a very heterogeneous group of tumors with alteration in SDH (SDH-deficient GIST accounting for approximately 20e40% of all KIT/PDGFRA nomutated GISTs), BRAF, NF1, KRAS genes or FGFR1::TACC1, ETV6::NTRK3 fusions. Methods: We examine the patient’s DNA and RNA using Sanger sequencing, HighResolution Melting, Allele-Specific PCR, and NGS methods to identify mutations of selected genes. Results: We detected primary mutations in KIT and PDGFRA genes in 84 % of samples (337 samples). The presence of secondary mutations we demonstrated in 17 patients (157 analyzed patients, 11 %) with the progression of the disease. We confirmed significant intratumor and also intertumor heterogeneity of secondary mutations. In the group of KIT/PDGFRA non-mutated GISTs (56 samples), we identify defects in the SDH complex subunits (12 samples, 21 %), mutations in the BRAF (2 samples, 3.5 %) and NF1 (1 sample, 2 %) genes, and alterations in the AKT1 (1 sample, 2 %) and ATR (1 sample, 2 %) genes. Conclusions: Determination of the GISTsmolecular subtype is very important considering therapeutic decisions in both the adjuvant and metastatic setting. The recent search for therapeutic variants is a significant molecular-genetic contribution to the framework of personalized medicine