BACKGROUND: Radiofrequency ablation (RFA) is an effective treatment for dysplastic Barrett's esophagus (BE), but recurrence can occur after initial response. Currently there is uncertainty about how to best define histological remission. A DNA methylation panel on esophageal samples was previously shown to have high diagnostic accuracy for BE. We aimed to investigate this biomarker panel in the assessment of response to RFA treatment. METHODS: We retrospectively analyzed esophageal and gastroesophageal junction (GEJ) biopsies from patients with BE before and after RFA treatment. We quantified the extent of intestinal metaplasia (IM) based on number of glands with goblet cells (IM-Score) and expression of the intestinal factor trefoil factor-3 (TFF3-Score). Promoter methylation of 3 genes (ZNF345, TFP12, ZNF569) was measured by methylight (Meth-Score) throughout the RFA treatment pathway. FINDINGS: We included 45 patients (11 non-dysplastic BE, 14 low-grade dysplasia, 20 high-grade dysplasia/intramucosal cancer). Meth-Scores were significantly higher in BE with and without dysplasia and GEJ with IM compared to GEJ without IM (P<·001). Meth-scores significantly correlated with the extent of IM at the GEJ measured both with IM-Scores (rho=66·0%, P<·001), and TFF3-Scores (rho=75·6%, P<·001). In patients with residual IM at the GEJ, RFA re-treatment brought about a 7·6-fold reduction in the methylation levels. The Meth-score had an area under the ROC curve of 95·1% (95%CI 91·1% - 99·1%) differentiating BE from normal GEJ. INTERPRETATION: A DNA methylation panel can discriminate between the extent of histological IM in esophageal and junctional biopsies and could be used to objectively quantify residual disease following RFA.