Background: During pregnancy, thyroid dysfunction has been associated with a number of adverse outcomes. The presence of anti- Thyroid Peroxidase Antibody (anti-TPO) also results in post-partum complications. Hypothyroidism is closely associated with the presence of anti-TPO. The study aimed to evaluate anti-TPO and thyroid function tests in first trimester of pregnancy. Materials and Methods: The study was carried out at Department of Biochemistry, Medical College and SSG Hospital, Vadodara, Gujarat, India over a period of one year after prior approval from institutional ethics committee. Total 200 normal pregnant women in their first trimester were randomly selected after informed written consent. Women having known thyroid dysfunction, other endocrinopathies, undergone thyroid surgery or taking thyroid medications were excluded. Overnight fasting blood samples from participants were analyzed for anti-TPO, thyroid stimulating hormone (TSH), total T3 and total T4. Results: Mean maternal age was 25.56 ± 3.32 years. Out of 200, total 30 (15%) pregnant women of first trimester had TSH level >2.5 mIU/l. Out of these 30 females, 9 (4.5%) had overt hypothyroidism & 21 (10.5%) had subclinical hypothyroidism. Among these 30 hypothyroid women, 26 (13%) had found anti-TPO positive (anti-TPO >35 IU//ml) which also showed a significant positive correlation with TSH level and negative correlation with T3 and T4 level. Conclusion: Hypothyroidism especially sub-clinical hypothyroidism is prevalent among pregnant women with positive correlation with anti-TPO. It is suggested that anti-TPO should be added as a screening test in first trimester of pregnancy which can be useful to identify early thyroid dysfunction