Objective To evaluate the accuracy of EUS, EUS-guided fine needle aspiration （EUSFNA） and targeted biopsy in the diagnosis of wall-thickened gastric lesions with negative malignant results of endoscopic biopsies. Methods A retrospective study was carried out in 57 patients who were found with thickened gastric wall of negative malignant endoscopic biopsies and underwent EUS from January 2008 to December 2010 in our hospital. Compared the EUS findings with the surgical results and follow-up status. The diagnostic yield of EUS was characterized by the disappearance of the layers or the changes of thickness of gastric wall, the characteristics of echo imaging, and the results of EUS-FNA or EUS targeted biopsy were recorded to evaluate the value of EUS. Results Of 57 cases, gastric cancer was confirmed in 19, lymphoma in 10, dysplasia in 1, Menetrier＇s disease in 1 and inflammatory changes in 26. EUS could clearly demonstrate the changes of gastric wall including the thickness and the changes of layers, with the accuracy rate of 73.07% （14/19） on gastric cancer. EUS diagnosed gastric cancer in 26 cases, in which 14 （53.8%） were confirmed by pathology. Gastric lymphoma was suspected by EUS in 20 cases, in which 10 （50.0%） were proved. EUS-FNA was conducted in 19 cases, with positive result in 9 （ accuracy rate 50% ）. EUS-guided targeted deep biopsy or piece-meal biopsy were performed in 10 cases, with 8 malignant results. Conclusion EUS with/without EUS-FNA is not a golden standard for the diagnosis of gastric lesions in thickened gastric wall, yet it still has some significance.