To investigate the effect of anisometropic and strabismic amblyopia on the nerve fiber layer thickness. This cross-sectional study was done on 54 amblyopic subjects, equally in both strabismic and anisometropic groups. The thickness otonerve fiber layer measured in superior, inferior, nasal, temporal quadrants and as a whole in both eyes of both groups. The means of thickness were compared in amblyopic and sound eyes. In strabismus group, the average nerve fiber layer thickness of the sound eye , in superior, inferior, nasal and temporal quadrants and as a whole were 113.23±14, 117.37±25, 68.96±6, 69.55±14 and 93.40±8 microns respectively. In amblyopic eyes of the same group, these measurements were 103.11±18, 67.74±11, and 69.59±16 and 89.59±12 microns in superior, inferior, nasal, temporal quadrants and as whole respectively. In anisometropic groups, the sound eye measurements were as 130.96±22, 129.07±29, 80.62±12, and 83.88±20 and 107.7±13 microns in superior, inferior, nasal and temporal quadrants and as a whole orderly. In amblyopic eyes of this group the mean thicknesses were 115.63±29, 133.15±25, 78.8±15, 80.2±16 and 109.17±21 microns in superior, inferior, nasal, temporal quadrants and as a whole respectively. Statistically, there were no significant differences between amblyopic and sound eyes (P>0.5). Our study did not support any significant change in a nerve fiber layer thickness of amblyopic patients; however, decreased thickness in superior and nasal quadrants of strabismic amblyopia and except inferior quadrant and as a whole. These measurements may be a clue for management and prognosis of amblyopia in old age