Gastrointestinal stromal tumors (GISTs) are characterized by overexpression and mutations of c-Kit. Approximately 80% of c-Kit mutations occur in exon 11, being a response factor to imatinib (Gleevec) therapy. We aimed to assess whether c-Kit and PDGFRA mutation analysis of GISTs obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) could be routinely performed. Mutation analysis of c-Kit hotspot exons (9, 11, 13 and 17) and PDGFRA hotspot exons (12 and 18) was performed in aspirates 51 mesenchymal tumors. We identified c-Kit mutations in 61% of GIST cases, in accordance with previously published ranges (30-90%). Nearly 95% (19/20) of c-kit-mutant tumors carried exon 11 mutations. Mutation analysis is possible in FNA cell blocks and can assist in the diagnosis and therapeutic decisions in GIST cases.