Epstein-Barr virus (EBV) is linked to the development of a variety of malignancies, including EBV-associated gastric carcinoma (EBVaGC). In this study, EBVaGC was detected in 15 (7.3%) of 206 GC cases. To identify the EBV genomic variation, EBV genomic sequences isolated from 9 EBVaGC biopsy specimens were successfully retrieved, designated EBVaGC1 to EBVaGC9. By comparative analysis of these strains with another 6 completely sequenced EBV strains, EBV-wild type, B95-8, AG876, GD1, GD2, and HKNPC1, it was demonstrated that EBVaGC1 to 9 were most closely related to the GD1 strain. Phylogenetic analysis of the GC biopsy specimen-derived EBV (GC-EBV) genomes was subsequently performed to assess their genomic diversity and it exhibited the greatest divergence from the type 2 strain, AG876. Compared with the reference EBV strain GD1, they harbored 961 variations in total, including 919 substitutions, 23 insertions, and 19 deletions. Single nucleotide polymorphism (SNP) density varied substantially across all known open reading frames and was highest in latency-associated genes. Moreover, we identified 2 interstrain recombinants at the EBNA1 locus, which provided a further mechanism for the generation of diversity. Some T-cell epitope sequences in EBNA1 and LMP2A genes showed extensive variation across strains, which implied their importance in the development of vaccines and T-cell therapy. In conclusion, we reported the first genome-wide view of sequence variation of EBV isolated from primary EBVaGC biopsy specimens, which might serve as an effective method for further understanding the genomic variations contribute to EBVaGC carcinogenesis and treatment.