Objective: To characterize the occurrence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its association with neonatal hyperbilirubinemia. Study design: This study involved an evaluation of G6PD data for 2656 newborns from a universal newborn screening program. Results: Mean G6PD activity was 14.2 ± 3.3 U/g Hb. Some 2.71% of the newborns were G6PD-deficient, and 1.77% had borderline G6PD activity, with male and female predominance, respectively. G6PD deficiency was more prevalent in newborns of Sephardic Jew and Muslim Arab backgrounds. The infants with G6PD deficiency had higher bilirubin levels at the time of discharge from the nursery. Infants with low and borderline G6PD activity were more likely to require phototherapy (22.2% and 25.5%, respectively, vs 7.6% of infants with normal G6PD activity; P <.005) and to have more referrals for exacerbation of jaundice (15.3% and 14.9%, respectively, vs 6.1%; P <.005). Mean G6PD activity was higher in preterm infants born at 27-34 weeks gestational age compared with those born later (16.3 ± 1.8 U/g Hb vs 14.8 ± 2.0 U/g Hb). Based on sex distribution and theoretical genetic calculations for the rate of heterozygous females, we propose that the range of borderline G6PD activity should be 2-10 U/g Hb rather than the currently accepted range of 2-7 U/g Hb. Conclusions: There is association between G6PD deficiency and significant neonatal hyperbilirubinemia. Increased risk is also associated with borderline G6PD activity. The suggested new range for borderline G6PD activity should enhance the identification of females at risk. G6PD activity is higher in preterm infants.