Purpose: To determine peripapillary retinal nerve fiber layer (RNFL) thickness values by Three-Dimensional optical coherence tomography (3D-OCT) in a normal Iranian Population and to evaluate the concordance of these measurements with those obtained by the second generation of optical coherence tomography (OCT II). Methods: In a cross-sectional observational study, 96 normal Iranian subjects 20-53 years old were enrolled. Peripapillary RNFL thickness in one randomly selected eye of each subject was measured by 3D-OCT and also by OCT II. Standard achromatic perimetry, corneal pachymetry and A-scan ultrasonographic biometry were also performed. Other study variables included age, gender, laterality (right versus left eye), refractive error, corneal diameter and disc area. Results: Mean peripapillary RNFL thickness measured by 3D-OCT (75.50±8.38) mm was significantly less than that measured by OCT II (144.10±33.32 mm) (P<0.001). Using 3DOCT, no significant difference in peripapillary RNFL thickness was observed by gender (P=0.90) or laterality (P=0.17); RNFL thickness had no correlation with age (P=0.95), axial length (P=0.32), spherical equivalent refractive error (P=0.21), central corneal thickness (P=0.66) and disc area (P=0.31). However, a positive correlation was found between peripapillary RNFL thickness and corneal diameter (P=0.03). Conclusion: 3D-OCT seems to yield lower RNFL thickness values as compared to OCT II. It seems advisable to obtain separate baseline measurements when using different generations of OCT machines.