Purpose: To investigate the diagnostic accuracy of Stratus optical coherence tomography (OCT) with its internal normative database to detect diffuse retinal nerve fiber layer (RNFL) atrophy in glaucoma subjects. Methods: One hundred two eyes of 102 glaucoma patients with diffuse RNFL atrophy and 102 healthy eyes of 102 age-matched subjects were enrolled in the Diffuse Atrophy Imaging Study. Two experienced observers graded RNFL photographs of diffuse RNFL atrophy eyes using a four-level grading system. The diagnostic performances for detecting diffuse RNFL atrophy were examined according to the visual field results as well as the RNFL photographs grading. Results: Using a criterion of abnormal at the < 5% level, the overall sensitivity of the Stratus OCT parameters ranged from 61.5% to 84.5%, and the overall specificity ranged from 90.2% to 99.0%. For mild, moderate and severe diffuse RNFL atrophy, the superior quadrant had a sensitivity of 41.0%, 83.3% and 100.0%, respectively, and the inferior quadrant had a sensitivity of 35.0%, 88.5% and 100.0%, respectively. The highest likelihood ratios were obtained at the 11 and 12 o'clock sectors for superior RNFL and 6 and 7 o'clock sectors for inferior RNFL. Conclusions: OCT with a normative database can detect diffuse RNFL atrophy with moderate sensitivity and high specificity. Because the sensitivity of Stratus OCT is closely related to the degree of diffuse RNFL atrophy as well as the visual field results, OCT with an internal normative database should be evaluated with prudence, especially in early stage of glaucoma with diffuse RNFL atrophy.