Background and Aims: Early esophageal squamous cell neoplasia (ESCN) is preferably treated with en-bloc endoscopic resection. Ablation might be an alternative for flat ESCN, but ESCN extension along the epithelial lining of ducts and submucosal glands (SMGs) might jeopardize ablation efficacy. Clinical studies suggest that local recurrence might arise from such buried ESCN niches after ablation. We studied human endoscopic resection specimens of ESCN to quantify ESCN extension into ducts/SMGs and performed a prospective porcine study to evaluate the depth of radiofrequency ablation (RFA) and CryoBalloon ablation (CBA) into ducts/SMGs. Methods: Endoscopic submucosal dissection specimens of flat-type ESCN from a Japanese (n = 65) and Dutch cohort (n = 14) were evaluated for presence and neoplastic involvement of ducts/SMGs. Twenty-seven pigs were treated with circumferential RFA (c-RFA; n = 4), focal CBA (n = 20), and focal RFA (n = 3) with 4, 60, and 9 treatment areas, respectively. After prespecified survival periods (0 hours, 8 hours, 2 days, 5 days, and 28 days), treatment areas were evaluated for uniformity and depth of ablation and affected SMGs. Results: Neoplastic extension in ducts/SMGs was observed in most lesions: 58% (38/65) in the Japanese and 64% (9/14) in the Dutch cohort. In the animal study, 33% of SMGs (95% confidence interval, 28-50) were not affected after c-RFA, although the overlying epithelium was ablated. Focal RFA and CBA resulted in uniform ablations with effective treatment of all SMGs. Conclusions: ESCN extends into ducts/SMGs in most patients. In an animal model, focal RFA and CBA effectively ablated SMGs, whereas c-RFA inadequately ablated SMGs. Given this potential reason for recurrence, endoscopic resection should remain the standard of care.