Introduction: The etiology of esophageal squamous papilloma (ESP) is largely unknown. Previous studies have shown a variable association with human papillomavirus (HPV) with conflicting data. The aim of this study was to further investigate the possible association of HPV in our ESP series using RNA in-situ hybridization (ISH) and compare study groups from the United States of America and China. Methods: Demographic and clinical data of patients with ESP were retrieved from the University of California Los Angeles (UCLA) (1/2016-3/2019) and Peking Union Medical College Hospital (PUMCH) (9/2014-3/2019) pathology databases. Hematoxylin and eosin slides were reexamined. Confirmed cases were examined by high- and low-risk HPV RNA ISH. Results: For the UCLA cohort, 13 429 upper endoscopies were performed and 78 biopsies from 72 patients were identified as ESP (F:M = 45:27, 66.7% > 45 years). Seventy-four (94.9%) biopsies were designated as polyps or nodules and 46.6% were located in the mid-esophagus. Other abnormal findings included gastroesophageal reflux disease (48.6%), hiatal hernia (38.9%), and esophagitis (36.1%). For the PUMCH cohort, 63 754 upper endoscopies were performed and 73 biopsies from 71 patients were identified as ESP (F:M = 48:23, 71.8% > 45 years). Sixty-four (87.7%) biopsies were designated as polyps or nodules and 57.5% were located in the mid-esophagus. Other abnormal findings included esophagitis (19.7%), and hiatal hernia (8.5%). No features of conventional cytologic dysplasia or viral cytopathic change were found. None of the cases was associated with squamous cell carcinoma, and none showed positive HPV RNA ISH results. Conclusions: No association was found between ESP and active HPV infection in our 2 cohorts. Other etiopathogenetic mechanisms, such as aging, might contribute to the development of these innocent lesions.