JC virus (JCV) is a neurotropic polyomavirus and the causative agent of progressive multifocal leukoencephalopathy. A role for JCV in gastrointestinal malignancies has been recently suggested. This study was carried out to determine the prevalence of polyomaviruses including JCV, BKV and SV40 in gastric cancers in Tunisia and to determine the clinicopathological characteristics of virus-associated gastric carcinomas. The presence of polyomaviruses DNA sequences was surveyed in 61 cases of primary gastric carcinomas and in 53 paired non-tumor gastric mucosa by PCR. Findings were correlated to clinicopathological parameters, p53 expression and methylation status of 11 tumor-related genes. Using PCR assays, JCV T-antigen sequence was more frequently detected in gastric carcinomas than in non-tumor gastric mucosa (26 vs 6%, P ¼ 0.03), while those of SV40 and BKV were not detected in any cases. Correlation analysis showed that JCV had higher frequency in patients older than 55 years (P ¼ 0.034) and in the intestinal histological type (P ¼ 0.04). With regard to methylation status, P16 and P14 showed significantly higher methylation frequencies in JCV-positive gastric carcinomas than in JCV-negative cases (P ¼ 0.007 and P ¼ 0.003, respectively). Moreover, the mean of the methylation index was significantly higher in JCV-positive than in JCV-negative cases (P ¼ 0.024). In multivariate logistic regression analysis, age of patients and the methylation index are only the two independent factors associated with JCV infection. Kaplan-Meier survival analysis showed a trend toward better survival for JCV-associated gastric carcinomas patients (log-rank, P ¼ 0.11). Our study suggests a role of JCV as cofactor in the pathogenesis of the intestinal type of gastric carcinomas in older persons.