Background: Several studies have examined association between human papillomaviruses (HPV) and esophageal cancer, but results have been inconsistent. This is the first prospective study to investigate associations between α, β and HPV detection in the oral cavity and risk of esophageal cancer. Methods: We conducted a nested case–control study among 96,650 cancer-free participants in the American Cancer Society Cancer Prevention Cohort and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Incident esophageal cancer cases ( n = 125) were identified during an average 3.9 years of follow-up. Three controls per case ( n = 372) were selected and matched on age, sex, race/ethnicity, and time since mouthwash collection. α, β, and HPV DNA in oral samples were detected using a next-generation sequencing assay. Conditional logistic regression models were used to estimate OR and 95% confidence intervals (CIs), adjusting for smoking and alcohol consumption. Statistical significance was evaluated using permutation test. Results: Prevalence of oral α, β, and HPV was 18.4%, 64.8%, and 42.4% in cases and 14.3%, 55.1%, and 33.6% in controls, respectively. Oral HPV16 detection was not associated with esophageal cancer (OR = 0.54, 95% CI, 0.1–4.84) and none of the esophageal squamous cell carcinoma cases ( n = 28) were HPV16 positive. Some oral HPV types were more common in cases than controls; however, none of the associations were statistically significant. Conclusions: Although HPVs in the oral cavity are very common, this study showed no evidence of association between oral HPVs and esophageal cancer. Impact: Oral HPVs may not contribute to risk of esophageal cancer. Cancer Epidemiol Biomarkers Prev; 27(10); 1168–75. ©2018 AACR .