Abstract Purpose: Human papillomavirus (HPV) has been well established as a causative factor in mucosal oropharyngeal cancer and anogenital surface malignancies. However, its etiological role in cutaneous squamous cell carcinoma (cSCC) is still debated. The aim of this study is to identify the presence of HPV types in cSCC and identify their associations with cSCC. Methods: 77 cSCC blocks from 54 patients underwent DNA isolation and a broad spectrum of HPV PCR tests to detect the presence of HPV genotypes by the use of a universal primer pair GP5+/GP6+. The resultant PCR products were cloned to T Vectors and subsequently underwent DNA sequencing and Nucleotide BLAST search to identify the HPV types in cSCC. To ensure the accuracy of the HPV PCR test, 16 blocks from 10 head and neck SCC (oropharyngeal) patients were also included in the HPV presence and type analyses. Cutaneous SCC patients’ demographics, pathology and clinical parameters were compared and analyzed statistically in conjunction with the HPV test results. Results: HPV DNA was found in 37 of 77 (48.1%) blocks representing 32 of 54 (59.3%) cSCC patients. HPV16 was the preponderant type of HPV Infection, accounting for 28 of 32 (87.5%) and 30 of 37 (81.08%) HPV positive patients and blocks. Other HPV genotypes found included HPV18, 57, 10, 2, accounting for 4 of 32 (12.5%), 2 of 32 (6.25%), 2 of 32 (6.25%), 2 of 32 (6.25%) of the HPV positive patients and 4 of 37 (10.81%), 2 of 37 (5.41%), 2 of 37 (5.41%), 2 of 37 (5.41%) of the HPV positive blocks, respectively. HPV infection was significantly associated with the cSCC tumor size in this cohort of patients. Tumors with a size of ≤20mm were more frequently associated with HPV. Moreover, the average tumor size of the HPV positive group was also significantly smaller than that of the HPV negative group. Conclusions: HPV infection is very commonly found in cSCC patients and HPV16 is the major type of HPV involved in cSCC oncogenesis, accounting for >80% of HPV infections. HPV is a contributing factor, which appears to be contributing to a smaller size of cSCC, which may improve the prognostic classification of patients to provide a new insight into tumorigenesis. Citation Format: Gang Shi, Anne C. Fischer, Jonathan M. Zenilman, Patti E. Gravitt, Max Fischer, Inbal B. Sander, Jonathan D. Cuda, Janis M. Taube, Mohammed Lilo, W P Andrew Lee, Anthony P. Tufaro. HPV16 was the preponderant type of HPV infection in cutaneous squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 674. doi:10.1158/1538-7445.AM2017-674