Background: It has been reported that tumor heterogeneity is frequently found in various types of human carcinomas. Tumor heterogeneity may exist in not only human tumors but also cancer cell lines. Analysis of tumor heterogeneity might be useful to understand the mechanisms of tumor development. In this study, we examined the heterogeneity of gastric cancer using a gastric cancer cell line and human gastric tumor samples. Materials and Methods: A gastric cancer cell line OCUM-12 and a total of 530 gastric cancers were used in this study. The heterogeneity of cancer cell line was examined after subcloning of OCUM-12 cells using limiting dilution. mRNA expression levels of TGF-ꞵ1 and TGF-ꞵ2 in each subclone cell were examined by RT-PCR. Next, we performed immunohistochemical study of TGF-ꞵ using 530 gastric cancers specimens. TGF-ꞵ expression was evaluated both at cancer cells and at cancer-associated fibroblasts (CAFs). Immunohistochemical staining level was evaluated by not only manual but also using digital QuPath. Correlation between TGF-ꞵ1 expression and clinicopathological features were analyzed. Results: mRNA expression levels of TGF-ꞵ1 and TGF-ꞵ2 was different among 12 OCUM-12 subclones. Immunohistochemical staining level of TGF-beta was also different among tumor specimens. Five-year recurrence-free survival rate of high TGF-beta expression group tended to be a poor prognosis (p = 0.0913) in Stage 2, while the survival rate showed a better prognosis in Stage 3. Conclusion: TGF-beta heterogeneity in cancer cells might be associated with tumor progression in gastric cancer.