Purpose To determine the prevalence of amblyopia in Denmark before and after the initiation of the Danish national preschool vision screening programme. Methods In a population-based cross-sectional study, 3826 participants of the Danish General Suburban Population Study (GESUS) aged 20 years and older from a Danish rural municipality received a complete general health examination and an ophthalmological interview and examination. This study included a comprehensive ophthalmologic interview, measurement of best corrected visual acuity (BCVA) in each eye, Hirschberg's test for strabismus and two 45-degree retinal fundus photographs of each eye. A complete ophthalmologic examination was performed when indicated. Results The prevalence of monocular visual impairment (MVI) was 4.26% (95% CI, 3.66-4.95, n = 163). Amblyopia was the most common cause, accounting for 33%. The prevalence of amblyopia was 1.44% (95% CI, 1.01-1.81, n = 55), being higher among non-preschool vision screened persons compared to those who were offered (estimated 95% attendance) preschool vision screening (1.78%, n = 41, 95% CI 1.24-2.33 versus 0.44%, n = 2, 95% CI, 0.12-1.60, p = 0.024). The leading cause of amblyopia was anisometropia (45.5%, 25/55). Conclusions Amblyopia was the most common cause of MVI. Following the initiation of the Danish national preschool vision screening programme, which has an approximate attendance rate of 95%, the prevalence of amblyopia decreased by fourfold.