We identified clinical and laboratory variables affecting the relationship between transcutaneous and serum bilirubin levels and determined whether transcutaneous bilirubin values could be used to predict the risk of hyperbilirubinemia. Median bias between transcutaneous and diazo serum bilirubin was 2.0 mg/dL (34.2 micromol/L), while median bias between transcutaneous and the Vitros (Ortho Clinical Diagnostics, Rochester, NY) serum bilirubin values was 1.3 mg/dL (22.2 micromol/L). The mother's ethnicity, the gestational age, and postnatal age did not impact the relationship between transcutaneous and serum bilirubin values. In contrast, the serum bilirubin method (diazo vs Vitros) and collection container (clear vs amber tube) significantly impacted the relationship between transcutaneous and serum bilirubin values. Transcutaneous bilirubin was a sensitive but not specific predictor of the risk of hyperbilirubinemia using a conventional risk nomogram. Because systematic differences between serum bilirubin methods and local laboratory practices impact the relationship between transcutaneous and serum bilirubin values, the effectiveness of transcutaneous prediction of the serum bilirubin risk zone will vary by institution.