Objective The purpose of this study was to evaluate the possible structural changes of the macula in patients with unilateral amblyopia using optical coherence tomography (OCT) image segmentation. Patients and Methods 38 consecutive patients (16 male; mean age 32.4±17.6 years; range 6–67 years) with unilateral amblyopia were involved in this study. OCT examinations were performed with a time-domain OCT device, and a custom-built OCT image analysis software (OCTRIMA) was used for OCT image segmentation. The axial length (AL) was measured by a LenStar LS 900 device. Macular layer thickness, AL and manifest spherical equivalent refraction (MRSE) of the amblyopic eye were compared to that of the fellow eye. We studied if the type of amblyopia (strabismus without anisometropia, anisometropia without strabismus, strabismus with anisometropia) had any influence on macular layer thickness values. Results There was significant difference between the amblyopic and fellow eyes in MRSE and AL in all subgroups. Comparing the amblyopic and fellow eyes, we found a statistically significant difference only in the thickness of the outer nuclear layer in the central region using linear mixed model analysis keeping AL and age under control (p = 0.032). There was no significant difference in interocular difference in the thickness of any macular layers between the subgroups with one-way between-groups ANCOVA while statistically controlling for interocular difference in AL and age. Conclusions According to our results there are subtle changes in amblyopic eyes affecting the outer nuclear layer of the fovea suggesting the possible involvement of the photoreceptors. However, further studies are warranted to support this hypothesis.