Barrett's oesophagus (BE) is a pre-malignant metaplastic tissue predisposing to oesophageal adenocarcinoma (EC), and gastro-oesophageal reflux is a risk factor for both conditions. Reflux of acid and bile can cause mucosal injury and initiate chronic inflammation. These processes can induce DNA damage, possibly via an oxidative stress mechanism, thus increasing the likelihood of progression from Barrett's metaplasia to dysplasia and finally carcinoma. The comet assay was optimized for the detection of DNA damage (strand breaks and alkali-labile sites) in oesophageal biopsies, including incorporation of the DNA repair enzyme Fapy-DNA glycosylase (Fpg). Fpg allows the detection of 8-hydroxy-2-deoxyguanosine (8-OHdG) sites, a known pro-mutagenic DNA lesion. BE patients were recruited from BE surveillance clinics and oesophageal biopsies collected at endoscopy. Comet analysis revealed significantly increased (p < 0.001) DNA damage in Barrett's epithelium compared with matched squamous epithelium, with median % tail DNA values of 25.1% (first to third quartile 21.7-29.6%) and 18.6% (first to third quartile 16.9-21.4%), respectively. The median % tail DNA was up to 70% higher in the matched BE tissue compared with squamous epithelium from the same patient. Fpg sensitive sites were demonstrated in both tissue types at similar levels. The raised level of DNA damage in the premalignant BE may contribute to the accumulation of genetic alterations occurring during progression to EC. Understanding these underlying mechanisms provides a basis for cancer prevention strategies in BE patients.