Purpose: The effectiveness of preverbal orthoptic tests at age 6, 9, 14 and 24 months in population-wide screening was assessed. Methods: Two consecutive birth cohorts at 134 centres were compared. At general health screening visits, children born July–December 2011 were vision screened four times between 6 and 24 months with inspection, pupillary reflexes, eye motility, Hirschberg, cover test and monocular pursuit. Children born January–June 2012 were vision screened at general screening visits only in case of visually apparent abnormalities or positive family history. After referral, cause and severity of amblyopia were determined. Visual acuity was measured in all children at 36 and 45 months. Results: The control and intervention group comprised 5649 versus 5162 children. Amblyopia was diagnosed in 185 (3.3%) versus 159 children (3.1%), outside of screening in 21 (11.4%) versus 25 (15.7%). Between 6 and 24 months, 44 (23.8%) versus 27 (17%) (RR = 0.67 [95% CI 0.42, 1.09]) were referred and after visual acuity (VA) measurement 120 (64.9%) versus 107 (67.3%). Of 109 versus 108 children with refractive or bilateral amblyopia, 94 (86.2%) versus 92 (85.2%) were detected with VA measurements. Visual acuity of the amblyopic eye, after referral, was not significantly different between groups (p 0.896), nor was the time to amblyopia diagnosis (intention to screen [p 0.55]; per protocol [p 0.11]). Conclusion: The effectiveness of vision screening was not influenced by omission of orthoptic tests at general health screening at 6–24 months. Refractive and bilateral amblyopia were almost exclusively found by VA measurements.