Analysis of a two-screen program for congenital hypothyroidism (CH) using differential blood TSH (bTSH) cutoffs of 10 mU/L at first screening (all infants) and 5 mU/L at second screening (selected infants).To characterize CH infants identified by the second screening and compare infants with bTSH 5.0-9.9 and ≥10 mU/L on second screening.Maternal and neonatal clinical features were retrospectively analyzed in 119 CH babies detected on the second screen in Lombardy region of Italy, 2007-2014.Fifty-two (43.7%) of the 119 CH neonates showed bTSH values ranging 5.0-9.9 mU/L at the second screening (Low bTSH Group) and 67 (56.3%) bTSH ≥10.0 mU/L (High bTSH Group). The frequency of thyroid dysgenesis and eutopic gland was similar in both groups, as was the frequency of permanent and transient CH. Moreover, a high frequency of extra-thyroidal malformations was found in both groups. The percentage of preterm infants (57.7% vs 23.9%, P<0.001) and infants admitted to NICU (50.0% vs 17.9%, P<0.001) was significantly higher in the Low versus the High bTSH Group. In addition, maternal treatment with glucocorticoids in pregnancy was significantly more frequent in the Low bTSH Group than in the High bTSH Group (11.5% vs 1.5%, P=0.042), as well as maternal hypothyroidism and/or goiter (26.9% vs 10.4%, P=0.036).This study has demonstrated that a lower TSH cutoff at the second screening can detect additional cases of CH and that a second bTSH cutoff of 5.0 mU/L is appropriate for identifying preterm newborns and babies with associated risk factors.