PURPOSE: To compare the peripapillary retinal nerve fiber layer (RNFL) thickness of sound and amblyopic eyes. DESIGN: Prospective observational case series. METHODS: SETTING: Institutional. STUDY POPULATION: Patients with unilateral strabismic, anisometropic, or combined amblyopia. OBSERVATION: Fast RNFL analysis with ocular coherence tomography (OCT) of sound and amblyopic eyes. MEASURE: Mean RNFL thickness. RESULTS: For the 17 patients (mean age 10.7 years) in whom both eyes were imaged, the mean thickness of the sound eye was 109.2 mu m (median 112.7) and of the amblyopic eye was 104.2 mu m (median 105.0), and the average difference (sound eye less amblyopic eye) was 5.0 mu m (median 3.0) (95% confidence interval -2.3, 12.2, P = .17). The sound eye was 10 mu m or more thicker than the amblyopic eye in four patients; the amblyopic eye was 10 mu m or more thicker than the sound eye in one patient; and the difference was within 10 mu m in 12 patients. Test-retest data were obtained for 23 pairs of sound eyes and 21 pairs of amblyopic eyes, with 75% of the test-retest pairs within 7%. CONCLUSIONS: We found a small, but not clinically significant, difference in nerve fiber layer (NFL) thickness between amblyopic and sound eyes. Reliability was excellent, with most eyes testing within 7% of the first test.