The aim of this study was to use a large group of observers to test prior research findings that suggest optic disc size, clinical evaluation of the neuroretinal rim (ISNT rule), and practitioner characteristics influence the accuracy of differentiating normal from glaucomatous optic nerves. Participant observers were optometrists, optometry students, and vision scientists/researchers attending the 2013 American Academy of Optometry Annual Meeting. Each observer viewed and judged six sets of stereoscopic photographs of normal and clinically confirmed glaucomatous optic nerves of different sizes presented in random order. Observers were queried on whether each nerve was glaucomatous or normal, whether the nerve followed the ISNT rule, and whether further evaluation with advanced imaging techniques was indicated. Of the 261 observers who participated, 59% were practicing optometrists, 7% were vision scientists, and 34% were residents or students. Of practicing optometrists and vision scientists, half (49%) had more than 15 years of experience, whereas 11% had less than 2 years of experience. Diagnostic accuracy differed based on optic nerve size: average-sized nerves were correctly identified by 90% of subjects, whereas small nerves and large nerves were correctly identified by 42% and 62%, respectively. Notably, only 9% of subjects correctly identified the small glaucomatous nerve, and only 34% correctly identified the large normal nerve. No practitioner characteristics were associated with diagnostic accuracy. Accurate identification of glaucomatous optic neuropathy was significantly influenced by optic disc size. This was particularly evident for the large normal nerve and the small glaucomatous nerve. The ISNT rule provided value for differentiating normal from glaucomatous nerves, but its subjective interpretation resulted in considerable intergrader variability. These findings agree with other studies utilizing smaller numbers of observers but larger numbers of optic nerve presentations that disc size and the ISNT rule have value for enhancing accuracy of optic nerve assessment.