Aims: To evaluate the prevalence of congenital hypothyroidism (CH) in a screening program performed for the first time in Isfahan, Iran. Methods: From May 2002 to December 2002, T<sub>4</sub> and TSH serum concentrations of 20,000 3- to 7-day-old newborns, born in all 17 hospitals of the city, were measured by radioimmunoassay and immunoradiometric assay, respectively. The newborns with abnormal screening results (TSH >20 mIU/l, T<sub>4</sub> <6.5 µg/dl and based on the weight) were re-examined. Results: Of 531 recalled subjects (recall rate 2.6%), 54 were confirmed to be hypothyroid, showing a prevalence of 1:370 for CH. Conclusion: Considering the high frequency of CH, the necessity of implementing a routine screening program in the healthcare system of Isfahan Province is emphasized.