<b><i>Introduction:</i></b> As preterm infants are susceptible to hyperbilirubinemia, they require frequent close monitoring. Prior to initiation of phototherapy, hour-specific total serum bilirubin (TSB) percentile cut-points are lacking in these infants, which led to the current study. <b><i>Methods:</i></b> A multi-site retrospective cohort study of preterm infants born between January 2013 and June 2017 was completed at 3 NICUs in Ontario, Canada. A total of 2,549 infants born at 29<sup>0/7</sup>–35<sup>6/7</sup> weeks’ gestation contributed 6,143 pre-treatment TSB levels. Hour-specific TSB percentiles were generated using quantile regression, further described by degree of prematurity, and among those who subsequently received phototherapy. <b><i>Results:</i></b> Among all infants, at birth, hour-specific pre-treatment, TSB percentiles were 36.1 µmol/L (95% confidence interval [CI]: 34.3–39.3) at the 40th, 52.3 µmol/L (49.4–55.1) at the 75th, and 79.5 µmol/L (72.1–89.6) at the 95th percentiles. The corresponding percentiles were 39.3 μmol/L (35.9–43.2), 55.4 μmol/L (52.1–60.2), and 87.1 μmol/L (CI 70.5–102.4) prior to initiating phototherapy and 24.4 μmol/L (20.4–28.8), 35.3 μmol/L (31.1–41.5), and 52.0 μmol/L (46.1–62.4) among those who did not receive phototherapy. Among infants born at 29–32 weeks, pre-treatment TSB percentiles were 53.9 µmol/L (49.4–61.0) and 95.5 µmol/L (77.5–105.0) at the 75th and 95th percentiles, with respective values of 48.7 µmol/L (43.0–52.3), and 74.1 µmol/L (64.8–83.2) for those born at 33–35 weeks’ gestation. <b><i>Conclusion:</i></b> Hour-specific TSB percentiles, derived from a novel nomogram, may inform how bilirubin is described in preterm newborns. Further research of pre-treatment TSB levels is required before clinical consideration.