Objective: To characterize differences in the prenatal diagnosis of birth defects (BDs) associated with singleton and multiple pregnancies. Design: An observational study conducted using data from a BDs surveillance system. Setting: Eastern China. Population: A total of 54,572 babies with BDs, born in 90 hospitals located in 30 regions of Zhejiang Province between 2012 and 2018. Methods: BDs were diagnosed by obstetricians using ultra sonographic, genetic tests et al., on the basis of the ICD 10th Revision (Q00–Q99). Main outcome measures: Differences in incidence and characteristics of BDs associated with singleton and multiple pregnancies. Multivariate logistic regression models were constructed to characterize relationships between the prenatal diagnosis of BDs and multiple pregnancies, with adjustment for covariates. Results: Totals of 49,872 singletons and 3,324 multiple pregnancies with BDs were analyzed. The mean incidences of BD for single and multiple pregnancies were 26.29 and 109.99 per 1,000 births, respectively (χ2=7600, P<0.001). After adjustment for covariates, BDs associated with multiple pregnancies were less likely to be diagnosed prenatally (adjusted OR: 0.36, 95% CI: 0.32–0.40); as were congenital heart defects, congenital hydrocephalus, cleft lip with cleft palate, congenital talipes equinovarus, cleft lip without cleft palate, limb reduction defects, congenital diaphragmatic hernia, trisomy 21 syndrome, congenital malformation of the urinary system, and other chromosomal malformation, compared with singletons with BDs. Conclusions: Multiple pregnancy is associated with a significantly higher risk of BD, but a significantly lower prenatal diagnosis rate. Therefore, the healthcare of women with multiple pregnancy and their fetuses should be strengthened.