【Objective】To study the expression of EB virus gene in head，neck and chest squamous cell carcinoma（HNCSCC），revealing the clinicopathological significance.【Methods】Using in situ hybridization and S-P immunohistochemical technique，we examined the expression of EBV and Ki67 protein in 123 cases of HNCSCC. The pathological characteristics such as sex ，age，histologic grade，lymph node metastasis，distant metastasis，situation and size were analyzed retrospectively.【Results】The positive expression rate of EBV in HNCSCC group was significantly higher than that in the normal mucosal epithelial group（P = 0.007）. EBV positive HNCSCC in female patients was 19.2%，markedly higher than 3.1%in male patients（χ2   = 8.781，P = 0.011）. EBV positive HNCSCC in the group under 50 years old was 18.8%（6/32），significantly higher than 2.2% in the group over 50 years old（χ2  = 10.666，P = 0.004）. The infection rate of EBV was 24.2% in 5 ~ 10 lymph nodes metastasis groups ，significantly greater than other groups 0%（Z = -3.548， P = 0.000）. EBV infection rate in the group without distant organ metastasis was 22.2% ，higher than 3.8% with distant organ  metastasis（χ2   =  8.567，P  =  0.016）.  The  group  with  the  Ki67  proliferation  index  less  than  70%  showed  higher positivity of EBers than the group with Ki67 more than 70%（8/76 vs. 0/47，Z = -2.291，P = 0.022）. There were not any statistically significant differences in histological differentiation，tumor situation and tumor diameter between EBV positive and negative groups. Regression analysis showed that age below 50 years old，the Ki67 index less than 70% and the more lymph node metastasis was elevated risk factors for EBV infection.【Conclusion】EBV infection is associated with HNC⁃ SCC. EBV-associated HNCSCC is more frequent in female patients under 50 years of age with more lymph node metasta⁃ sis，while distant metastasis is less common.