BACKGROUND AND STUDY AIMS: Diffuse infiltrative gastric cancer can be difficult to diagnose owing to a lack of endoscopic features in the superficial mucosa. Moreover, a forceps biopsy may not reveal a pathological diagnosis. We aimed to evaluate the diagnostic yield and safety of endoscopic mucosal resection (EMR) and 'open-lid submucosal biopsy', a technique wherein EMR followed by biopsy of the ulcer floor is performed for a pathological diagnosis. PATIENTS AND METHODS: This retrospective observational study involved patients with suspected diffuse invasive gastric cancer on endoscopy and cross-sectional imaging in whom endoscopic forceps biopsy did not lead to a pathological diagnosis. Patients who underwent EMR and open-lid submucosal biopsy were included. The primary outcome was the total diagnostic yield. The secondary outcomes were the diagnostic yields of EMR and open-lid submucosal biopsy and incidence of complications. RESULTS: Between June 2011 and February 2022, EMR and open-lid submucosal biopsy for diagnostic purposes were performed on seven patients without complications. EMR was diagnostic in four (57%) cases of diffuse infiltrative gastric cancer. Open-lid submucosal biopsy was diagnostic in five (71%) cases. As the primary endpoint, the combination of EMR and open-lid submucosal biopsy led to a pathological diagnosis in six cases (86%). Secondary analysis revealed gastric adenocarcinoma in 9 of 22 (41%) EMR specimens, while open-lid submucosal biopsies were positive for adenocarcinoma in 14 of 60 specimens (23%). CONCLUSIONS: The combination of EMR and open-lid submucosal biopsy was safe and showed good accuracy for the diagnosis of diffuse infiltrative gastric cancer.