Purpose. This study was conducted to determine the thickness map of eleven retinal layers in normal subjects by spectral domain optical coherence tomography (SD-OCT) and evaluate their association with sex and age. Methods. Mean regional retinal thickness of 11 retinal layers were obtained by automatic three-dimensional diffusion-map-based method in 112 normal eyes of 76 Iranian subjects. Results. The thickness map of central foveal area in layer 1, 3, and 4 displayed the minimum thickness (P<0.005 for all). Maximum thickness was observed in nasal to the fovea of layer 1 (P<0.001) and in a circular pattern in the parafoveal retinal area of layers 2, 3 and 4 and in central foveal area of layer 6 (P<0.001). Temporal and inferior quadrants of the total retinal thickness and most of other quadrants of layer 1 were significantly greater in the men than in the women. Surrounding eight sectors of total retinal thickness and a limited number of sectors in layer 1 and 4 significantly correlated with age. Conclusion. SD-OCT demonstrated the three-dimensional thickness distribution of retinal layers in normal eyes. Thickness of layers varied with sex and age and in different sectors. These variables should be considered while evaluating macular thickness.