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Sepsis in the first 3 days of life is a leading cause of morbidity and mortality among infants. Group B Streptococcus (GBS), historically the primary cause of early-onset sepsis (EOS), has declined through widespread use of intrapartum chemoprophylaxis. We estimated the national burden of invasive EOS cases and deaths in the era of GBS prevention.Population-based surveillance for invasive EOS was conducted in 4 of the Centers for Disease Control and Prevention's Active Bacterial Core surveillance sites from 2005 to 2008. We calculated incidence using state and national live birth files. Estimates of the national number of cases and deaths were calculated, standardizing by race and gestational age.Active Bacterial Core surveillance identified 658 cases of EOS; 72 (10.9%) were fatal. Overall incidence remained stable during the 3 years (2005: 0.77 cases/1000 live births; 2008: 0.76 cases/1000 live births). GBS (∼38%) was the most commonly reported pathogen followed by Escherichia coli (∼24%). Black preterm infants had the highest incidence (5.14 cases/1000 live births) and case fatality (24.4%). Nonblack term infants had the lowest incidence (0.40 cases/1000 live births) and case fatality (1.6%). The estimated national annual burden of EOS was approximately 3320 cases (95% confidence interval [CI]: 3060–3580), including 390 deaths (95% CI: 300–490). Among preterm infants, 1570 cases (95% CI: 1400–1770; 47.3% of the overall) and 360 deaths (95% CI: 280–460; 92.3% of the overall) occurred annually.The burden of invasive EOS remains substantial in the era of GBS prevention and disproportionately affects preterm and black infants. Identification of strategies to prevent preterm births is needed to reduce the neonatal sepsis burden.