Objective To screen the newborns with glucose-6-phosphate dehydrogenase (G6PD) deficiency in Shanghai, determine the cut off value for the screening laboratory, reduce the false positive rate, and improve the quality of population. Methods G6PD levels of filter paper blood samples were detected by the quantitative fluorometric method. Definite diagnoses were made for suspected cases by the G6PD/6PGD quantitative ratio method. Results A total of 217 447 newborns (116

549 boys and 100 898 girls) were screened and 270 of them were definitely diagnosed with G6PD deficiency (250 boys and 20 girls). The incidences of G6PD deficiency of all newborns, boys, and girls were 0.18%, 0.31%, and 0.03%, respectively. The cut off values of G6PD of all newborns, boys, and girls were 2.165 U/g, 1.97 U/g, and 2.165 U/g, respectively. Conclusion Early diagnosis and prevention of G6PD deficiency can be achieved by the neonatal screening, which should be popularized in Shanghai. Screening laboratories should determine the cut off values for newborns, boys, and girls that can reduce the false positive rate for screening G6PD deficiency and improve the screening efficiency.