The aim of this study was to investigate the agreement between cycloplegic and non-cycloplegic autorefraction with an open-field auto refractor in a school vision screening set up, and to define a threshold for myopia that agrees with the standard cycloplegic refraction threshold. The study was conducted as part of the Sankara Nethralaya Tamil Nadu Essilor Myopia (STEM) study, which investigated the prevalence, incidence, and risk factors for myopia among children in South India. Children from two schools aged 5 to 15 years, with no ocular abnormalities and whose parents gave informed consent for cycloplegic refraction were included in the study. All the children underwent visual acuity assessment (Pocket Vision Screener, Elite school of Optometry, India), followed by non-cycloplegic and cycloplegic (1% tropicamide) open-field autorefraction (Grand Seiko, WAM-5500). A total of 387 children were included in the study, of whom 201 were boys. The mean (SD) age of the children was 12.2 (±2.1) years. Overall, the mean difference between cycloplegic and non-cycloplegic spherical equivalent (SE) open-field autorefraction measures was 0.34 D (limits of agreement (LOA), 1.06 D to −0.38 D). For myopes, the mean difference between cycloplegic and non-cycloplegic SE was 0.13 D (LOA, 0.63D to −0.36D). The prevalence of myopia was 12% (95% CI, 8% to 15%) using the threshold of cycloplegic SE ≤ −0.50 D, and was 14% (95% CI, 11% to 17%) with SE ≤ −0.50 D using non-cycloplegic refraction. When myopia was defined as SE of ≤−0.75 D under non-cycloplegic conditions, there was no difference between cycloplegic and non-cycloplegic open-field autorefraction prevalence estimates (12%; 95% CI, 8% to 15%; p = 1.00). Overall, non-cycloplegic refraction underestimates hyperopia and overestimates myopia; but for subjects with myopia, this difference is minimal and not clinically significant. A threshold of SE ≤ −0.75 D agrees well for the estimation of myopia prevalence among children when using non-cycloplegic refraction and is comparable with the standard definition of cycloplegic myopic refraction of SE ≤ −0.50 D.