Background: We aimed to evaluate the protein and mRNA expression of fibroblast growth factor receptor 2 (FGFR2) by immunohistochemistry (IHC) and mRNA in situ hybridization (ISH), respectively, and to assess the heterogeneity of FGFR2 expression in gastric cancer (GC). Methods: A tissue microarray containing 362 surgically resected GC tissues and 135 matched metastatic lymph nodes was evaluated using FGFR2b IHC and FGFR2 ISH. FGFR2 fluorescence ISH was also performed in 188 cases. Results: All FGFR2-amplified cases (5 of 188) showed FGFR2b protein and FGFR2 mRNA overexpression (p < 0.001), and FGFR2 amplification was not identified in FGFR2b IHC- and FGFR2 mRNA ISH-negative cases. Kaplan-Meier survival analysis revealed that FGFR2b protein and FGFR2 mRNA overexpression was significantly associated with a poor overall survival (p < 0.001 and p = 0.012, respectively), and multivariate analyses showed that FGFR2 mRNA overexpression was an independent biomarker of a poor overall survival. Intratumoral heterogeneity of FGFR2b protein and FGFR2 mRNA overexpression was observed in 5 of 9 (55.5%) and 18 of 21 (85.7%) cases, respectively. Discordant FGFR2b and FGFR2 expression results between primary and matched metastatic lymph nodes were observed in 5 of 9 (55.5%) and 4 of 14 (28.6%) cases, respectively. Conclusions: Intratumoral heterogeneity and discordant FGFR2b expression in primary tumors and metastatic lymph nodes are common in GC.