Background RTVue spectral-domain optical coherence tomography (OCT) is a new, ultra high-speed and high-resolution instrument, potentially to measure the presence of glaucoma or its progression accurately. The objective of this study was to evaluate its reproducibility of retinal nerve fiber layer (RNFL) thickness and optic nerve head (ONH) measurements in normal and glaucoma eyes. Methods This study was an observational clinical study. One eye was selected randomly from each of 89 normal individuals and 63 glaucoma patients in a range of severity. RNFL thickness and ONH were measured 3 times on the same day to determine intrasession variability. The same instrument was used by the same operator for all scans. Intrasession within-subject standard deviation (Sw), precision (1.96×Sw), coefficient of variation (CVw, 100×Sw/overall mean), and intraclass coefficient (ICC) were calculated to evaluate reproducibility.Results RTVue OCT demonstrated double hump patts in the RNFL profiles. High reproducibility was observed in all ONH parameters. For normal eyes, the value of ICC ranged between 0.98 and 1.00. For eyes with different extent of glaucoma, it ranged between 0.94 and 1.00. High reproducibility was also observed in RNFL thickness measurements.The values of ICC for averaged RNFL thickness ranged between 0.95 and 1.00 in all cases. For regional parameters, it ranged from 0.94 to 0.98 for normal eyes, 0.94 to 1.00 for mild glaucoma eyes, 0.87 to 1.00 for moderate glaucoma eyes, and 0.77 to 0.97 for severe glaucoma eyes. The nasal regions of severe glaucoma appeared to be most variable, as nasal lower region and inferior nasal region had the ICC values of 0.77 and 0.87. Conclusion Reproducibility of RTVue RNFL and ONH measurements was excellent in normal and glaucoma groups.