Purpose: To assess the prevalence and severity of developmental defects of enamel (DDE) in primary teeth and maternal-associated factors. Methods: This cross-sectional study included 336 two- to four-year-old children who attended the National Day of Children's Vaccination in São Paulo State, Brazil. The modified DDE index was used for diagnosis. Mothers completed sociodemographic and health questionnaires. Descriptive and Poisson regression analyses were performed. Results: The prevalence of DDE was 50.6 percent. The most frequent defects were demarcated opacities (45.0 percent), diffuse (36.0 percent) opacities, and hypoplasia (5.8 percent). White opacities were predominant (64.8 percent) in the teeth with defects, followed by cream (20.4 percent), yellow (5.2 percent), and brown (1.9 percent). Most defects involved less than one-third of the tooth surface (80.2 percent). The prevalence of DDE was associated with maternal-child factors such as alcohol consumption during pregnancy (prevalence ratio [PR] equals 1.27; 95 percent confidence interval [95% CI] equals 1.03 to 1.55), child hospitalization for infectious disease in the first year of life (PR equals 1.32; 95% CI equals 1.05 to 1.67), and breastfeeding for the first 12 months of life (PR equals 0.53; 95% CI equals 0.45 to 0.62). Conclusions: Developmental defects of enamel showed high prevalence and mild severity in the primary dentition. Alcohol consumption during pregnancy and child hospitalization for infectious diseases in the first year of life were associated with an increased prevalence of DDE. Moreover, children who breastfed for 12 months had a lower prevalence of DDE in primary teeth.