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Background: Epstein-Barr virus (EBV) is detected in epithelial tumors, such as nasopharyngeal carcinoma and gastric cancer (GC). EBV-associated gastric cancer is a distinct molecular subtype of gastrointestinal carcinomas as defined by Cancer Genome Atlas. This gamma-Herpes virus is present in approximately 10% of gastric carcinomas.Methods: The present study aimed to investigate the presence of EBV genome in gastric cancer and other gastroduodenal diseases either alone or together with Helicobacter pylori (HP). We examined 237 samples from Iranian patients diagnosed with GC and gastroduodenal disease for EBV infection by quantitative Real-Time PCR.Results: Of the 237 samples tested, EBV DNA was detected in 37 samples (15.6%), in 13 of the 81 GC cases (16%), and 24 of the 156 non-cancerous samples (15.4%). All samples containing EBV were gastric cancer of the intestinal type. Of 37 EBV-positive samples, 20 (54.1%) were over 55 years old and 20 (54.1%) were male. The EBV-EBER (EBV-encoded small RNA) DNA copy number in the gastric cancer group (mean = 2.14×10-1 copies/cell) was higher than that in the gastroduodenal disease group (mean = 1.39×10-2 copies/cell), and this difference was statistically significant (P> 0.001). Moreover, the concurrent infections with EBV and HP were detected in 17 out of 35 EBER-positive samples (48.6%), 6 (17/1%) cases were in the gastric cancer group and 11 (31/4%) cases were in the gastroduodenal disease group.Conclusions: In the present study, a high incidence (16%) of EBVaGC was observed in Babol city, Northern Iran. Also, the higher number of copies of EBV EBER DNA in the GC group than in the non-cancer group confirmed the possible role of EBV in inducing cancer. EBVaGC is not endemic in any region and varies in different nations. Therefore, further studies are needed to determine the role of this virus in the development of GC and other gastroduodenal diseases.