This prospective study was performed to measure the macular and the peripapillary retinal nerve fiber layer (RNFL) thicknesses using optical coherence tomography (OCT) in patients with anisometropic amblyopia. Thirty-one patients with hyperopic anisometropic amblyopia were included. The macular retinal thickness and the peripapillary RNFL thickness were measured using OCT. The mean refractive error was +3.71 diopters (D) and +1.00 D, the mean macular retinal thickness was 252.5 microm and 249.7 microm, and the mean RNFL thickness was 115.2 microm and 109.6 microm, inthe amblyopic eye and the normal eye, respectively. OCT assessment of RNFL thickness revealed a significantly thicker RNFL in hyperopic anisometropic amblyopia (P=0.019), but no statistically significant difference was found in macular retinal thickness (P>0.05). In conclusion, the amblyopic process may involve the peripapillary RNFL, but not the macula. However, further evaluation is needed.