Epstein-Barr virus (EBV) is associated with Burkitt lymphoma, nasopharyngeal carcinoma, opportunistic lymphomas in immunocompromised hosts, and a fraction of gastric cancers. Aberrant promoter methylation accompanies human gastric carcinogenesis, though the contribution of EBV to such somatic methylation changes has not been fully clarified. We analyzed promoter methylation in gastric cancer cases with Illumina's Infinium BeadArray and used hierarchical clustering analysis to classify gastric cancers into 3 subgroups: EBV(-)/low methylation, EBV(-)/high methylation, and EBV(+)/high methylation. The 3 epigenotypes were characterized by 3 groups of genes: genes methylated specifically in the EBV(+) tumors (e.g., CXXC4, TIMP2, and PLXND1), genes methylated both in EBV(+) and EBV(-)/high tumors (e.g., COL9A2, EYA1, and ZNF365), and genes methylated in all of the gastric cancers (e.g., AMPH, SORCS3, and AJAP1). Polycomb repressive complex (PRC) target genes in embryonic stem cells were significantly enriched among EBV(-)/high-methylation genes and commonly methylated gastric cancer genes (P = 2 × 10(-15) and 2 × 10(-34), respectively), but not among EBV(+) tumor-specific methylation genes (P = 0.2), suggesting a different cause for EBV(+)-associated de novo methylation. When recombinant EBV was introduced into the EBV(-)/low-methylation epigenotype gastric cancer cell, MKN7, 3 independently established subclones displayed increases in DNA methylation. The promoters targeted by methylation were mostly shared among the 3 subclones, and the new methylation changes caused gene repression. In summary, DNA methylation profiling classified gastric cancer into 3 epigenotypes, and EBV(+) gastric cancers showed distinct methylation patterns likely attributable to EBV infection.