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It is controversial that retina and photoreceptors are involved in amblyopic development. Lack of imaging devices for quantitative assessment of photoreceptors in vivo impedes further investigation. In this study we investigated the differences of macular thickness, outer nuclear layer (ONL) thickness and cone density among eyes with unilateral anisometropic amblyopia, amblyopic fellow eyes, and normal control eyes using high-definition spectral-domain optical coherence tomography and adaptive optics imaging system. Thicker fovea and parafovea with characteristic quantitative retinal changes in unilateral amblyopic patients indicated that the amblyopic process might involve the retina. Cones tended to be homogeneously distributed at 1.5° from the foveal center in all participants. However, we did not detect any significant differences in cone density and ONL thickness at 1.5° from the foveal center in patients with unilateral amblyopia when compared with the fellow eyes and the normal control eyes. This is the first study to explore whether photoreceptors are involved in amblyopic development in vivo that would help for understanding the underlying mechanisms of amblyopia. Whether macular changes relate to cone photoreceptors migration of the amblyopic eyes remains to be determined.