To determine the frequency of unilateral amblyopia in right versus left eyes among children younger than 18 years.Analysis of data collected in randomized clinical trials conducted by the Pediatric Eye Disease Investigator Group.The laterality of the amblyopic eye was analyzed in 2635 subjects younger than 18 years who participated in 9 multicenter prospective, randomized treatment trials. Eligibility criteria for these clinical trials included unilateral amblyopia associated with strabismus, anisometropia, or both, with visual acuity between 20/40 and 20/400. Logistic regression was used to assess the association of baseline and demographic factors with the laterality of amblyopia.Among subjects with anisometropic amblyopia (with or without strabismus), amblyopia was present more often in left than right eyes, with a relative prevalence of 59% in left eyes (95% confidence interval, 57% to 62%; P < .001 from a test of proportion, 50%). However, among subjects with strabismic-only amblyopia, there was no laterality predilection (relative prevalence of 50% in left eyes; 95% confidence interval, 47% to 54%; P = .94).Anisometropic amblyopia, with or without strabismus, occurs more often in left eyes than right eyes. This finding of amblyopia laterality may be related to microtropia, sighting dominance, or other forms of ocular dominance; developmental or neurological factors; laterality in the development of refractive error; or a combination thereof.