Barrett's esophagus (BE) with high-grade dysplasia is generally treated by endoscopic mucosal resection or esophagectomy. Radiofrequency ablation (RFA) is a recent treatment that allows broad and superficial ablation for BE. Endoscopic three-dimensional optical coherence tomography (3D-OCT) is a volumetric imaging technique that is uniquely suited for follow-up surveillance of RFA treatment. 3D-OCT uses a thin fiberoptic imaging catheter placed down the working channel of a conventional endoscope. 3D-OCT enables en face and cross-sectional evaluation of the esophagus for detection of residual BE, neo-squamous mucosa, or buried BE glands. Patients who had undergone RFA treatment with the BARRX HALO90 system were recruited and imaged with endoscopic 3D-OCT before and after (3-25 months) RFA treatment. 3D-OCT findings were compared to pinch biopsy to confirm the presence or absence of squamous epithelium or buried BE glands following RFA. Gastric, BE, and squamous epithelium were readily distinguished from 3D-OCT over a large volumetric field of view (8mmx20mmx1.6 mm) with ∼5μm axial resolution. In all patients, neosquamous epithelium (NSE) was observed in regions previously treated with RFA. A small number of isolated glands were found buried beneath the regenerated NSE and lamina propria. NSE is a marker of successful ablative therapy, while buried glands may have malignant potential and are difficult to detect using conventional video endoscopy and random biopsy. Buried glands were not observed with pinch biopsy due to their extremely sparse distribution. These results indicate a potential benefit of endoscopic 3D-OCT for follow-up assessment of ablative treatments for BE.