Recent research has revealed that genetic defects due to mutation in the Thyroid Peroxidase (TPO) gene can lead to thyroid dysfunction in the population. We aimed to study the association between genetic defects in TPO gene and patients with hypothyroidism found in adult age. Two hundred consecutive treatment naive hypothyroid patients (age >= 18 years) (cases) who were negative for anti TPO antibody and their corresponding sex and age matched two hundred normal individuals (controls) were enrolled. The 17 exonic regions of the TPO gene were amplified and sequenced directly. We identified 6 different previously known single nucleotide polymorphisms (SNPs) and 2 novel deletions in TPO gene. Two of the six SNPs revealed a significant association with hypothyroidism; Thr725Pro (rs732609) and Asp666Asp (rs1126797). The c.2173C allele of the Thr725Pro in TPO showed a significant association among hypothyroid patients compared to controls (p = 0.01; Odds ratio=1.45; 95% CI: 1.09-1.92) suggesting it to be a potential risk allele toward disease predisposition. Analysis of genotype frequencies of the polymorphism between the two groups demonstrated CC as a potential risk genotype (p = 0.006; Odds ratio=1.95; 95% CI: 1.2-3.15) for the disease while another SNP Asp666Asp (c.1998T allele) showed protectiveness towards the disease (p = 0.006; Odds ratio = 0.67; 95%CI: 0.50-0.89). To our knowledge, this is first study reporting the role of TPO gene with hypothyroidism in a population of Asian Indian origin. The study threw up the possibility of TPO gene polymorphisms as a possible pathogenetic mechanism of hypothyroidism.