Background:Reference intervals for thyroid hormone during pregnancy need to be gestational age, method, and population specific and there is need to establish trimester-specific thyroid levels for the different population across the world. The aim of this study was to establish trimester-specific reference range for thyroid hormone during pregnancy in a tertiary care center in Haryana.Materials and Methods:A total of 1430 pregnant women were recruited for the study. Participants having any history of chronic illness, goiter on physical examination, thyroid illness in the past or present, consuming thyroid medications, family history of thyroid illness, presence of anti-thyroid peroxidase antibody, poor obstetrics history were excluded from the study and reference population was identified to calculate serum free triiodothyronine (FT3), free thyroxine (FT4) and thyrotropin (TSH) for each trimester of pregnancy.Results:The 2.5–97.5th percentiles for FT3, FT4, and TSH obtained in this study were 2.53–4.54 pg/ml, 0.88–1.78 ng/ml and 0.37–3.69 μIU/ml in the first trimester, 2.0–4.73 pg/ml, 0.91–1.78 ng/ml and 0.54–4.47 μIU/ml in the second trimester, 2.01–4.01 pg/ml, 0.83–1.73 ng/ml, and 0.70–4.64 μIU/ml in the third trimester of pregnancy. Mean TSH increased and mean FT3 decreased significantly with the progression of gestational period. FT4 decreased from trimester 1–3rd, but the decrease was nonsignificant from 2nd to 3rd trimester.Conclusions:Existing results for trimester-specific reference intervals for thyroid hormones are inconsistent and cannot be extrapolated due to differences in ethnicity, maternal iodine status, laboratory assay method, and rigor for selection of reference population. Thus, establishment of reference intervals in each region is of great importance.