Background: Universal screening of congenital cytomegalovirus (cCMV) infection is important for monitoring and intervention during critical stages of speech and language development. This study aimed to explore the optimal detection strategy for cCMV infection screening.Methods: Serum samples from pregnant women and saliva and urine samples from their newborns were collected for the anti-CMV IgG and CMV DNA PCR tests, respectively. The sensitivity, specificity, and predictive values as well as the likelihood ratios of 12 potential screening strategies for cCMV infection, based on tests for saliva, urine, and their combination, were evaluated.Findings: A total of 6729 pregnant women were enrolled, and the seroprevalence was 98.1%. Among 6350 newborns that were followed up, 49 were defined as having cCMV infection. In the screening test, the CMV DNA positivity rate remained similar from day 0 to day 5, increased slowly from day 6 to day 13, and became high in newborns beyond 13 days of birth. In the confirmatory testing, the positive rates increased significantly beyond day 21. For the 49 newborns with cCMV infection, the proportion of agreement between saliva and urine testing was poor. Upon evaluating alternative screening strategies, using saliva and urine screening with saliva and urine confirmation as the reference strategy, saliva screening with saliva and urine confirmation showed good diagnostic accuracy and feasibility, with sensitivity, specificity, positive predictive and negative predictive values of 85.7%, 100.0%, 100.0% and 99.9%, respectively.Interpretation: In populations with high seroprevalence, saliva screening with saliva and urine confirmation might be an alternative strategy for screening cCMV infections. The suggested timeframes for screening and confirmation are within 13 (ideally 5) and 21 (ideally 13) days of birth, respectively.