BackgroundExtremely preterm infants are prone to hyperbilirubinemia and its sequelae. Currently recommended thresholds for initiating phototherapy in these newborns are consensus-based (CB).MethodsA multi-site retrospective cohort study of 642 infants born at 240/7 to 286/7 weeks’ gestation, between January 2013 and June 2017, was conducted at three NICUs in Canada. Pre-phototherapy TSB percentile levels at 24 h of age were generated and contrasted with published CB thresholds.ResultsAmong infants born 240/7 to 256/7 weeks’ gestation, the differences between our TSB percentiles vs. the CB threshold of 85.0 µmol/L were 10.0 µmol/L (95% CI, 6.0–16.0) at the 75th percentile and 35.3 µmol/L (95% CI, 26.1–42.8) at the 95th percentile. Respectively, among infants born at 260/7 to 276/7 weeks, differences were 19.4 µmol/L (95% CI, 16.8–23.4) and 43.3 µmol/L (95% CI, 34.7–46.9). Born at 280/7 to 286/7 weeks’ gestation, differences between our 75th and 95th TSB percentiles and the CB threshold of 103 µmol/L were 6.9 µmol/L (95% CI, 3.2–12.0) and 36.0 µmol/L (95% CI, 31.0–44.3), respectively.ConclusionsWe provide statistically derived pre-phototherapy TSB levels that may clarify patterns of pre-phototherapy TSB levels in extremely preterm infants.ImpactWe present statistically derived pre-phototherapy total serum bilirubin levels in a cohort of extremely preterm infants.Most of these preterm infants received phototherapy—some at below currently published thresholds.There are notable differences between our statistically derived pre-phototherapy TSB levels and currently published lower limit TSB thresholds for phototherapy.Our study results assist in the understanding of pre-phototherapy TSB levels in extremely preterm infants.