During the first trimester of pregnancy, thyroid-stimulating hormone (TSH) >2.5 mIU/L has been suggested as the universal criterion for subclinical hypothyroidism. However, TSH levels change continuously during pregnancy, even in the first trimester. Therefore the use of a fixed cut-off value for TSH may result in a different diagnosis rate of subclinical hypothyroidism according to gestational age. The objective of this study was to obtain the normal reference range of TSH during the first trimester in Korean gravida and to determine the diagnosis rate of subclinical hypothyroidism using the fixed cut-off value (TSH >2.5 mIU/L). The study population consisted of pregnant women who were measured for TSH during the first trimester of pregnancy (n=492) and nonpregnant women (n=984). Median concentration of TSH in pregnant women was lower than in non-pregnant women. There was a continuous decrease of median TSH concentration during the first trimester of pregnancy (median TSH concentration: 1.82 mIU/L for 3+0 to 6+6 weeks; 1.53 mIU/L for 7+0 to 7+6 weeks; and 1.05 mIU/L for 8+0 to 13+6 weeks). Using the fixed cut-off value of TSH >2.5 mIU/L, the diagnosis rate of subclinical hypothyroidism decreased significantly according to the gestational age (GA) at TSH (25% in 3+0 to 6+6 weeks, 13% in 7+0 to 7+6 weeks, and 9% for 8+0 to 13+6 weeks, P<0.001), whereas the diagnosis rate was 5% in all GA with the use of a GA-specific cut-off value (P=0.995). Therefore, GA-specific criteria might be more appropriate for the diagnosis of subclinical hypothyroidism.