Basaloid squamous cell carcinoma (BSCC) of the esophagus is rare, historically confused for adenoid cystic carcinoma, and recently shown to behave similar to conventional, keratinizing esophageal squamous cell carcinoma. At other sites (eg, oropharynx, anogenital tract) the basaloid phenotype is frequently associated with the presence of high-risk human papillomavirus (HPV). HPVs role in esophageal squamous cell carcinomas is less certain, and to our knowledge, a direct examination of esophageal BSCC for high-risk HPV has not been performed earlier. Nine cases of esophageal BSCC were retrieved from our surgical pathology files. Twenty-two cases of keratinizing esophageal squamous cell carcinoma served as controls. In situ hybridization (ISH) for high-risk HPV and immunohistochemistry for related molecular markers including p53, cyclin D1, and p16 (scored 0 to 4+ based on percentage of cells staining; p53 additionally scored for intensity) were performed. HPV ISH was nonreactive in all tested cases. Compared with controls, BSCC showed less immunoreactivity for p16 and p53 (P=0.003, 0.009). Esophageal BSCC is negative for high-risk HPV by ISH, distinguishing these lesions from other BSCCs. Differential p16 and p53 expression in BSCC suggests that these tumors are molecularly distinct from conventional esophageal squamous cell carcinomas.