An association between viral infection, particularly the human papillomavirus, and the development of esophageal carcinoma (EC) has been reported. However, reports concerning the relationship between herpes simplex virus (HSV) and Epstein-Barr virus (EBV) with EC are few. There are geographic variations in infection rates. This study was aimed to determine the co-incidence of infection of the two viruses' with esophageal carcinoma and the differentiation of cancer tissues and lymphocytes infiltration in the tumor stroma of the high-incidence area of Shantou China. To determine the association between viral infection (HSV and EBV) and EC, we applied in situ hybridization (ISH) and immunohistochemistry (IHC) in 164 esophageal carcinoma surgical specimens from the high-incidence area of Shantou China. HSV DNA and HSVI, II protein expression were found in 52 (31.7%) of the 164 tumors; EBV EBER and LMP-1 proteins were identified in only 10 (6.1%) carcinoma specimens by in situ hybridization and immunohistochemistry. In histopathology analysis, the positive cases of HSV appeared to be more predominant in well and moderately differentiated squamous cell carcinomas, and the positive cases of EBV were found in poorly differentiated squamous cell carcinomas or undifferentiated carcinomas with intense lymphoid infiltration. Our results confirm the involvement of HSV and EBV in esophageal carcinomas and the relationship between HSV and EBV infection and esophageal carcinoma cell differentiation with lymphocyte infiltration in the tumor stroma. However, the two herpes viruses, HSV and EBV, particularly the human HSV may be one of the etiological factors in development of this malignancy among the high-incidence population of Shantou China.