Abstract: Objective To investigate the infection status and clinicopathological characteristics of Epstein-Barr virus（EBV）and human papillomavirus（HPV）in oral squamous cell carcinoma（OSCC）in Guangzhou,China. Methods A total of 304 OSCC specimens were collected from Guangzhou,including 123 cases from the Third Affiliated Hospital of Sun Yat-sen University（January 2014 to December 2024）and 181 cases from Sun Yat-sen Memorial Hospital of Sun Yat-sen University（January 2017 to December 2018）. Polymerase Chain Reaction（PCR）was used to detect viral DNA,in situ hybridization was used to assess EBV-encoded RNAs（EBER）,and immunohistochemistry was performed to identify viral-related proteins and determine the type of infection. Results Among the 304 OSCC specimens,26.3%（80/304）were EBV-PCR positive,with 63.8%（51/80）diagnosed at TNM stage I/II. Compared with EBV-negative cases,the difference in tumor staging was statistically significant（P = 0.042）. Among the EBV-PCR-positive OSCC cases,the EBER-positive rate was 33.8%（27/80）,with 92.6%（25/27）localized in the cytoplasm. The detection rates of EBV-encoded nuclear antigen-1（EBNA-1）,latent membrane protein-1（LMP-1）,and EBV-encoded nuclear antigen-2（EBNA-2）were 83.8%（67/80）,81.3%（65/80）,and 56.3%（45/80）,respectively,while the lytic protein ZEBRA was not detected. Type III latent infection accounted for 41.3%（33/80）. In contrast,HPV DNA was detected in only 2.6%（8/304）of OSCC cases,among which two were p16-IHC positive. Only one case of OSCC showed EBV-HPV coinfection. Conclusions Based on samples collected from Sun Yat-sen University affiliated hospitals,EBV infection is relatively prevalent in OSCC cases in Guangzhou,mainly characterized by type III latency and cytoplasmic localization of EBER,suggesting a potential role of EBV in the pathogenesis of OSCC. In contrast,HPV-related OSCC appears to be rare and likely plays a limited pathogenic role.