This study aimed at developing strategies for screening, predicting, and diagnosing intrauterine HBV infection in infants born to HBsAg positive mothers. A total of 1,360 infants born to 1,355 HBsAg positive mothers were followed for 1 year. All newborn infants received active and passive immunization within 24 hr after birth. Maternal and infant blood samples were collected and tested for the status of serum HBsAg, HBeAg, and HBV DNA positivity. The accuracy of infant HBsAg positivity, HBV DNA positivity, HBsAg and HBV DNA double positivity, and HBsAg and/or HBV DNA positivity at birth in the diagnosis of intrauterine HBV infection was evaluated by receiver operating characteristic curve analysis. Of 1,360 infants, 145 tested positive for HBsAg and/or HBV DNA at birth. Twenty-one (1.5%) infants, who were diagnosed with intrauterine HBV infection, showed HBsAg positivity from birth to 7 and 12 months of age. Infant HBsAg positivity at birth had the highest sensitivity in predicting intrauterine HBV infection, while neonatal HBsAg and HBV DNA double positivity had the highest specificity. These findings suggest that infants, who were born to HBsAg positive mothers and were positive for both HBsAg and HBV DNA at birth, may be at a higher risk for intrauterine HBV infection. HBsAg positivity at birth may be a good marker for screening intrauterine HBV infection. Infant HBsAg positivity both at birth and 7 months of age may be used as a diagnostic criterion to simplify diagnostic procedures and improve diagnostic efficiency.