Background: Submucosal lesion is a mass or bulge covered by normal-appearing mucosa identified during standard endoscopy. Endoscopic Ultrasound (EUS) allows precise differentiation of the individual layers of the gastrointestinal (GI) tract. Endoscopic Ultrasound-Fine Needle Aspiration (EUS-FNA) permits safe and accurate sampling of submucosal lesions (SMLs) for further histopathology. Gastrointestinal stromal tumors are the commonest mesenchymal neoplasms of the GI tract. They should be stratified by malignant potential according to tumor size, location, and mitotic count. Objectives: The aim of work was to evaluate the role of endoscopic ultrasound with EUS-FNA in management of submucosal gastrointestinal lesions, with special concern about gastrointestinal stromal tumors (GISTs) lesions and the criteria highly associated with malignant nature. Patients and methods: This multi-center prospective study included a total of 150 patients with gastrointestinal submucosal lesions, conducted to evaluate EUS and EUS-FNA as diagnostic tools of submucosal lesions, including 68 GIST lesions. All patients underwent conventional endoscopy, EUS-FNA and histopathological examination of the samples obtained in Theodor Bilharz Research Institute, Cairo University Hospital and Kobry El Qubba Military Hospital from 2015 to 2017. Results: There were 150 patients with submucosal GI lesions, including 93(62%) males and 57(38%) females, with the mean age of 52±11.9 years. Presentations included dyspepsia 73(48.7%), asymptomatic SMLs 31(20.7%). The lesions were mainly gastric 102(68%). Final diagnoses were GIST 68 (45.3%), lipoma 11(7.3%), leiomyoma 9(6%) and extramural lesions 7(4.7%). There were 68 patients with GIST lesions, including 43(63.2%) males and 25(36.8%) females, with the mean age of 54±10.3 years. Presentations included dyspepsia 41(63.2%), asymptomatic SMLs 16(23.5%). The lesions were mainly gastric 57(83.8%). Sensitivity and specificity were 88.6% and 100% of EUS-FNA as diagnostic tools for GIST lesions respectively. (PPV) and (NPV) were 100% and 82.8% respectively. Conclusion: It could be concluded that EUS and EUS-FNA were highly significant methods in diagnosis of GIST lesions in relation to final histopathology.