Background: Congenital hypothyroidism is a major but preventable cause of mental backwardness in infants. In case of hypothyroidism in fetuses, certain complications will occur in vital organs like the central nervous system and the skeleton. Before screening programs were introduced, congenital hypothyroidism was diagnosed with delay due to its few and non-specific symptoms during the first days of life. Given the difference in reference range for various races and populations, this study was aimed to determining a natural range for thyroid hormones used in the screening of infants. Methods: In a descriptive cross-sectional study at Vali-asr Hospital, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran, from March 2013 to 2014. The level of T4, FT4 and TSH hormones was measured in all natural childbirth, without any complications and with one-minute Apgar score above 7 while this study was underway. Results: A group of 249 infants (male and female), including 35 (14.1%) aged between 0 and 4 days, 102 (41%) aged between 5 and 7 days and 112 (45%) aged between 7 and 30 days, were examined. The average TSH, T4 and FT4 amounts in infants, aged 0 to 30 days, were respectively 5.35 µU/ml, 10.77 µg/dl and 1.33 ng/dl. The interval was 4.76-6.01 µU/ml for TSH, 10.36-11.17 µg/dl for T4 and 1.29-1.37ng/dl for FT4. The difference between age groups with average TSH concentration is not meaningful (P=0.7) and the average T4 amount in different age groups was meaningfully different (P=0.05). The average FT4 in different age groups is meaningfully different (P=0.007). The thyroid hormone amounts calculated for males and females were not significantly different. Conclusion: Given the difference in the reference range in race, population and geographical zones, it is necessary to determine a range for screening in Iran. According to the findings of the present study, lower cut-offs, compared with western countries, should be envisaged for TSH in congenital hypothyroidism screening programs that could speed up the diagnosis of minor cases of the disease and prevent complications.