Incidence of human papillomavirus (HPV)–associated oral cancers is on the rise. However, epidemiological data of this subset of cancers is limited. Dental hospital pose a unique advantage in detection of HPV-positive oral malignancies. We assessed the utility of formalin-fixed, paraffin-embedded (FFPE) tissues, which are readily available, for evaluation of high-risk HPV infection in oral cancer. For protocol standardization, we used 20 prospectively-collected, paired FFPE and fresh tissues of histopathologically-confirmed oral cancer cases reported in Oral Medicine department of a dental hospital for comparative study. Only short-PCRs (~200bp) of DNA isolated using a modified xylene-free method displayed a concordant HPV result. For HPV analysis, we used additional 30 retrospectively-collected FFPE tissues. DNA isolated from these specimens showed an overall 23.4% (11/47) HPV positivity with detection of HPV18. Comparison of HPV positivity from dental hospital FFPE-specimens with overall HPV positivity of freshly-collected oral cancer specimens (n-55) from three cancer care hospitals of the same region showed notable difference (12.7%; 7/55). Further, cancer hospital specimens showed HPV16 positivity and displayed a characteristic difference in reported sub-sites and patient spectrum. Overall, using a xylene-free FFPE-DNA isolation method clubbed with short amplicon PCR, we showed detection of HPV-positive oral cancer in dental hospitals.