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Background: Accurate measurement of retinal nerve fiber layer (RNFL) is now possible with the high resolution optical coherence tomography (OCT). Effect of refractive status of the eye on RNFL thickness may be relevant in the diagnosis of glaucoma and other optic nerve diseases. Aim: To assess the RNFL thickness and compare its correlation with refractive status and axial length of the eye. Material and Methods: Three hundred eyes of 150 patients were included in this study, who underwent RNFL analysis using TOPCON 3D OCT 2000. Analysis of variance has been used to find the significance of study parameters between the study groups. Results: The study showed that refractive status/axial length affected the peripapillary RNFL thickness significantly. Conclusion: The study suggests that the diagnostic accuracy of OCT may be improved by considering refractive status and axial length of the eye when RNFL is measured.