Nationally, newborn screening programs use 17-hydroxyprogesterone (17-OHP) as the biomarker to detect the rare but potentially fatal inherited disease, congenital adrenal hyperplasia. However, this biomarker is highly variable, with a high false-positive rate of detection, particularly in neonates born preterm. Several studies have examined various clinical and genetic factors to explain the variability of 17-OHP in preterm infants. The purpose of this study was to replicate previous clinical and genetic associations with 17-OHP in a well-characterized cohort of 762 preterm infants. We replicated previous findings that respiratory distress syndrome (p = 2 x 10(-3)) is associated with higher 17-OHP. Higher 17-OHP and false positives were significantly associated with lower gestational age and birth weight, as previously reported. Incorporating gestational age and birth weight together decreases the false-positive rate.