Aim: Genotyping and prevalence of human cytomegalovirus (HCMV) in gastrointestinal tract remains unclear. Objective: Characterization of HCMV and its viral load in samples of patients with gastric cancer (GC) in northeast Iran. Methods: A total of 21 biopsies were collected from patients with GC. HCMV was detected and genotyped using multiplex nested PCR. Quantitative real-time PCR has been used to determine the viral load. Staining was performed to detect viral inclusions. Results: 76.19% of the samples had HCMV genotypic b2 with viral load <0.1 IU/μl. 66.67% of patients were positive for H. pylori. Conclusion: Multiplex nested-PCR method can detect CMV populations with low viral loads in GC biopsies. For the first time, it was determined that the genotype b2 of HCMV is prevalent in the gastrointestinal tract.