Context: Antibodies against thyroid peroxidase (TPOAb) are detected in 90% of all patients with Hashimoto's thyroiditis, the most common cause of hypothyroidism. Hypothyroidism is associated with a range of adverse outcomes. The current knowledge of its genetic underpinnings is limited. Objective: To identify novel genetic variants associated with TPOAb concentrations and positivity using genome-wide association data, and to characterize their association with thyroid function and disease. Design, setting and participants: European ancestry participants of three independent prospective population-based studies: Atherosclerosis Risk In Communities study (n= 7524), Study of Health in Pomerania (n=3803) and SHIP-TREND (n=887). Exposure: Single nucleotide polymorphisms (SNPs), individually and combined into a genetic risk score (GRS). Main Outcomes: TPOAb concentrations and positivity, thyroid hormone concentrations (TSH, FT4), clinical thyroid diseases (subclinical and overt hypothyroidism, goiter) Results: Significantly associated SNPs (p<5*10(-8)) mapped into four genomic regions not previously implicated for TPOAb (RERE, extended HLA-region), and into five previously described loci. A higher GRS based on these nine SNPs showed strong and graded associations with higher TPOAb, TSH and lower FT4 concentrations (p<0.001). Compared to individuals in the lowest GRS quartile, those in the highest quartile had 1.80-fold higher odds of subclinical hypothyroidism (95% CI 1.27-2.55) and 1.89-fold higher odds of overt hypothyroidism (95% CI 1.24-2.87). Conclusion: The identification of four novel genetic loci associated with TPOAb concentrations and positivity furthers insight into the genetic underpinnings of hypothyroidism. A genetic risk score showed strong and graded associations with markers of thyroid function and disease in independent population-based studies.