Introduction To evaluate the relationship between retinal nerve fiber layer (RNFL) thickness measured by Cirrus high-definition (HD) optical coherence tomography (OCT) and the axial length and refractive error of the eye.Materials and methods A total of 100 eyes of 100 healthy subjects (age 20-34 years with M/F ratio of 57/43), comprising 50 eyes with emmetropia [spherical equivalent (SE) 0 D], 25 eyes with moderate myopia (SE between -4 D and -8 D), and 25 eyes with high myopia (SE between -8 D and -12 D) were analyzed in this cross-sectional study. Average and mean clock hour RNFL thicknesses were measured by cirrus HD-OCT and compared between the three groups. Associations between RNFL measurements and axial length and SE were evaluated by linear regression analysis.Results The average RNFL measurements were significantly lower in high myopia (78.68 +/- 5.67) and moderate myopia (83.76 +/- 3.44) group compared with emmetropia group (91.26 +/- 2.99), also in the superior and inferior mean clock hours. Significant correlations were evident between RNFL measurements and the SE and axial length. The average RNFL thickness decreased with increasing axial length (r = -0.8115) and negative refractive power (r = 0.8397). Myopia also affected the RNFL thickness distribution. As the axial length increased and the SE decreased, the thickness of the superior, inferior, and nasal peripapillary RNFL decreased.Conclusion The axial length/refractive error of the eye affected the average RNFL thickness and the RNFL thickness distribution. Analysis of RNFL thickness in the evaluation of glaucoma should always be interpreted with reference to the refractive status. When interpreting the RNFL thickness of highly myopic patients by OCT, careful attention must be given to the inherently thinner RNFL to avoid a false diagnosis of glaucoma.How to cite this article Singh D, Mishra SK, Agarwal E, Sharma R, Bhartiya S, Dada T. Assessment of Retinal Nerve Fiber Layer Changes by Cirrus High-definition Optical Coherence Tomography in Myopia. J Curr Glaucoma Pract 2017;11(2):52-57.