PURPOSE: To determine the prevalence of and factors associated with amblyopia in a sample of Australian preschool children. DESIGN: Population-based, cross-sectional study. PARTICIPANTS: The Sydney Paediatric Eye Disease Study examined 2461 (73.8% participation) children aged between 6 and 72 months from 2007 to 2009. METHODS: Visual acuity (VA) was assessed in children aged ≥ 30 months using the Electronic Visual Acuity system, and a subset using the logarithm of the minimum angle of resolution chart. Amblyopia was categorized into unilateral and bilateral subtypes: Unilateral amblyopia was defined as a 2-line difference in reduced VA between the 2 eyes, in addition to strabismus, anisometropia, and/or visual axis obstruction; bilateral amblyopia was defined as bilateral reduced VA with either bilateral visual axis obstruction or significant bilateral ametropia. Information on ethnicity, birth parameters, and measures of socioeconomic status were collected in questionnaires completed by parents. MAIN OUTCOME MEASURES: Amblyopia. RESULTS: We included 1422 children aged 30 to 72 months, of whom 27 (1.9%) were found to have amblyopia or suspected amblyopia. Mean spherical equivalent for the amblyopic eyes was +3.57 diopters, with a mean VA of 20/50. Only 3 of the 27 amblyopic children had previous diagnoses or treatments for amblyopia. In regression analysis controlling for age, gender, and ethnicity, amblyopia was significantly associated with hyperopia (odds ratio [OR], 15.3; 95% confidence interval [CI], 6.5-36.4), astigmatism (OR, 5.7; 95% CI, 2.5-12.7), anisometropia (OR, 27.8; 95% CI, 11.2-69.3), and strabismus (OR, 13.1; 95% CI, 4.3-40.4). There were no significant associations of amblyopia with low birthweight (<2500 g), preterm birth (<37 weeks), maternal smoking, age, gender, ethnicity, or measures of socioeconomic status (all P>0.05). CONCLUSIONS: Amblyopia was found in 1.9% of this Australian preschool sample, which is comparable with prevalence rates reported by other recent studies in preschool children. Refractive errors, particularly significant hyperopia and astigmatism, in addition to anisometropia and strabismus, were the major amblyogenic factors. There was a low amblyopia detection rate in this preschool population, which suggests that different strategies are required to improve current vision screening strategies in preschoolers. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any of the materials discussed in this article.