Background Late-onset sepsis (LOS) is an important cause of death and neurodevelopmental impairment in premature infants. The purpose of this study was to assess overall incidence of LOS, distribution of LOS-causative organisms, and center variation in incidence of LOS for extremely premature infants over time. Methods In a retrospective analysis of infants 401–1000 g birth weight and 22–28 6/7 weeks’ gestational age born at 12 NICHD Neonatal Research Network centers in the years 2000–2005 (Era 1) or 2006–2011 (Era 2) who survived >72 hours, we compared the incidence of LOS and pathogen distribution in the 2 eras using the chi-square test. We also examined the effect of birth year on the incidence of LOS using multivariable regression to adjust for non-modifiable risk factors and for center. To assess whether the incidence of LOS was different among centers in Era 2, we used a multivariable regression model to adjust for non-modifiable risk factors. Results 10,131 infants were studied. LOS occurred in 2083/5031 (41%) infants in Era 1 and 1728/5100 (34%) infants in Era 2 (P<.001). Birth year was a significant predictor of LOS on adjusted analysis, with birth years 2000–2009 having a significantly higher odds of LOS than the reference year 2011. Pathogens did not differ, with the exception of decreased fungal infection (P<.001). In Era 2, nine centers had significantly higher odds of LOS compared with the center with the lowest incidence. Conclusions The incidence of LOS decreased over time. Further investigation is warranted to determine which interventions have the greatest impact on infection rates.