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Abstract: Background: Barrett’s esophagus is a condition that is premalignant for adenocarcinoma of the esophagus and the esophagogastric junction. Early detection of Barrett’s metaplasia and dysplasia is very important to decrease the mortality and morbidity from esophageal adenocarcinoma cancer. This study aimed to evaluate the effectiveness of methylene blue–targeted biopsies in the differential diagnosis of intestinal metaplasia, dysplasia, and superficial esophageal carcinoma. Methods: A total of 109 patients (43 women and 66 men; average age, 62.32 ± 10.61 years; range, 33–82 years) were enrolled for the study. Four groups were designed before endoscopic examinations. The patients for these groups were selected at the conventional endoscopy, and then chromoendoscopy was performed. The esophagus was stained with methylene blue, after which six biopsies were taken from stained and unstained areas. Results: Conventional and chromoendoscopic assessments were compared with histopathologic examination. The sensitivity of chromoendoscopy for Barrett’s epithelium was superior to that of conventional endoscopy (p < 0.05). However, there was no statistical difference between the two methods in the diagnosis of esophagitis or esophageal carcinoma (p > 0.05). Stained biopsies were superior to unstained biopsies in terms of sensitivity for Barrett’s epithelium and esophageal carcinoma (p < 0.001). Conclusion: Chromoendoscopy is useful for delineating Barrett’s epithelium and for indicating the correct location for securing biopsies where dysplasia or early esophageal cancer is suspected.