Background: Radiofrequency ablation (RFA) is effective for treating Barrett's esophagus (BE) but often involves multiple endoscopy sessions over several months to achieve complete response. Objective: Identify structural markers that correlate with treatment response by using 3-dimensional (3-D) optical coherence tomography (OCT; 3-D OCT). Design: Cross-sectional. Setting: Single teaching hospital. Patients: Thirty-three patients, 32 male and 1 female, with short-segment (<3 cm) BE undergoing RFA treatment. Intervention: Patients were treated with focal RFA, and 3-D OCT was performed at the gastroesophageal junction before and immediately after the RFA treatment. Patients were re-examined with standard endoscopy 6 to 8 weeks later and had biopsies to rule out BE if not visibly evident. Main Outcome Measurements: The thickness of BE epithelium before RFA and the presence of residual gland-like structures immediately after RFA were determined by using 3-D OCT. The presence of BE at follow-up was assessed endoscopically. Results: BE mucosa was significantly thinner in patients who achieved complete eradication of intestinal metaplasia than in patients who did not achieve complete eradication of intestinal metaplasia at follow-up (257 ± 60 μm vs 403 ± 86 μm; P <.0001). A threshold thickness of 333 μm derived from receiver operating characteristic curves corresponded to a 92.3% sensitivity, 85% specificity, and 87.9% accuracy in predicting the presence of BE at follow-up. The presence of OCT-visible glands immediately after RFA also correlated with the presence of residual BE at follow-up (83.3% sensitivity, 95% specificity, 90.6% accuracy). Limitations: Single center, cross-sectional study in which only patients with short-segment BE were examined. Conclusion: Three-dimensional OCT assessment of BE thickness and residual glands during RFA sessions correlated with treatment response. Three-dimensional OCT may predict responses to RFA or aid in making real-time RFA retreatment decisions in the future.