Oxygen saturation imaging is a new technology that determines biological features from the perspective of oxygen concentration. Therefore, this exploratory study aimed to evaluate the biological implications of oxygen saturation imaging and further assess tumor heterogeneity in gastric cancer. Biopsy samples were selectively obtained from treatment-naïve patients with gastric cancer under real-time oxygen saturation imaging. Tissue oxygen saturation level calculations, immunohistochemistry, and RNA sequencing were performed. The mean tissue oxygen saturation levels at the sampling sites were 32.2%, 70.8%, and 56.2% for hypoxic, hyperoxic, and non-tumor areas, respectively, with significant differences between each pair. CD-31 and glucose transporter 1 protein expression were significantly upregulated in hypoxic tumors. Comprehensive transcriptomic analysis revealed enriched biological processes related to the regulation of insulin-like growth factor transport and uptake by insulin-like growth factor-binding proteins in hypoxic tumors and the type I interferon signaling pathway in hyperoxic tumors. Oxygen saturation imaging has the potential to clarify hypoxia-induced heterogeneity in gastric cancer from both clinical and fundamental perspectives.