Background: Endoscopic surveillance of pre-malignant gastric lesions may add to gastric cancer prevention. However, the appropriate biopsy regimen for optimal detection of the most advanced lesions remains to be determined. Therefore, we evaluated the yield of endoscopic surveillance by standardized and targeted biopsy protocols. Materials and Methods: In a prospective, multi-center study, patients with intestinal metaplasia (IM) or dysplasia (DYS) underwent a surveillance gastroscopy. Both targeted biopsies from macroscopic lesions and 12 non-targeted biopsies according to a standardized protocol (antrum, angulus, corpus, cardia) were obtained. Appropriate biopsy locations and the yield of targeted versus non-targeted biopsies were evaluated. Results: In total, 112 patients with IM (n = 101), or low-grade (n = 5) and high-grade DYS (n = 6) were included. Diagnosis at surveillance endoscopy was atrophic gastritis (AG) in one, IM in 77, low-grade DYS in two, high-grade DYS in three, and gastric cancer in one patient. The angulus (40%), antrum (35%) and lesser curvature of the corpus (33%) showed the highest prevalence of pre-malignant conditions. Non-targeted biopsies from the lesser curvature had a significantly higher yield as compared to the greater curvature of the corpus in diagnosing AG and IM (p = .05 and p = .03). Patients with extensive intragastric IM, which was also present at the cardia were at high risk of a concurrent diagnosis of dysplasia or gastric cancer. High-grade DYS was detected in targeted biopsies only. Conclusions: At surveillance endoscopies, both targeted and non-targeted biopsies are required for an appropriate diagnosis of (pre-)malignant gastric lesions. Non-targeted biopsies should be obtained in particular from the antrum, angulus and lesser curvature of the corpus.