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Objective: To detect retinal nerve fiber layer thickness differences of ambylopic and fellow eyes and ambylopic and control eyes. Methods: The study comprised a total of 152 eyes recruited from Erzurum Region Training and Research Hospital, Turkey, between January 2018 and May 2018. Anisometropic amblyopia was the only cause of disability (visual acuity ≤ 6/12 and a difference in best-corrected visual acuity (BCVA) between the two eyes of 0.20 logMAR (2 lines on an acuity chart) in amblyopic eyes (n, 74) whereas normal eyes had a best-corrected visual acuity of 6/6 and no morbidities (n, 78). Anisometropic patients were divided into three groups as hyperopic, myopic, and cylindiric. All amblyopic eyes were compared with fellow eye and control group. Retinal nerve fiber layer thickness was analyzed using optic coherence tomography (OCT) (RTVue 100-2, Optovue, Inc. Fremont, CA). Results: The mean age of the patients was 28.64 ± 8.23 years in amblyopia group and 32.23 ± 8.14 years in control group (p, 0.008). Mean best-corrected visual acuity (BCVA) was 0.36 ± 0.23 in amblyopic eyes, 0.96 ± 0.15 in fellow eyes, and 1.00 ± 0 in control group. Mean refractive error was 2.76 ± 7.84 in amblyopic eyes, 0.42 ± 1.34 in fellow eyes, and 0.12 ± 0.27 in control group. Temporal retinal nerve fiber layer thickness (RNFL-T) was 77.27 ± 10.38 μ, 79.31 ± 9.53 μ, and 81.46 ± 9.86 μ respectively in ambylopic group, fellow eyes, and control eyes (p, 0.036). Superior RNFL-T was 136.23 ± 18.52 μ, 131.91 ± 13.80 μ, and 135.56 ± 14.94 μ respectively in ambylopic group, fellow eyes, and control eyes (p, 0.204). Nasal RNFL-T was 87.99 ± 13.05 μ, 82.16 ± 12.33 μ, and 85.50 ± 10.62 μ respectively in ambylopic group, fellow eyes, and control eyes (p, 0.014). Inferior RNFL-T was 144.85 ± 18.39 μ, 140.55 ± 16.92 μ, and 143.47 ± 17.75 μ respectively in ambylopic group, fellow eyes, and control eyes (p, 0.322). There was statistically significant difference in temporal and nasal quadrants and no statistical difference in superior and inferior quadrants. Discussion: The presence of amblyopia seems not to be related with RNFL-T so we could ignore anisometropic amblyopia in patients with disease that could be detected and followed via RNFL thickness. Further and larger scaled studies are needed for certain results. [ABSTRACT FROM AUTHOR]