Background: The thyroid gland constitutes an integral component of the endocrine system, exerting regulatory control over numerous vital physiological processes through the synthesis and release of specific hormones. Hypothyroidism is a disorder in which the thyroid gland fails to produce enough thyroid hormone to maintain normal physiological function. The prevalence of hypothyroidism varies substantially worldwide and is more common in populations with a relatively high iodine intake or severe iodine deficiency, Hashimoto's thyroiditis, inflammation of the thyroid, congenital hypothyroidism, hypothyroidism that is present from birth, iodine deficiency or excess, as well as diseases of the pituitary gland or hypothalamus, are the most frequent causes of hypothyroidism. Objective: The study aims to investigate the relationship between some vitamins and hypothyroidism in Iraqi patients. Subjects and Methods: This is a case-control study, manipulated from the first of March to the end May of 2023, 90 samples aged (18-70) years old, from Baghdad City, were selected and divided them into two groups. The first group consists of 60 people infected with hypothyroidism. The second group (control group) consists of 30 apparently healthy people. Blood samples were assessed for serum vitamin B12, and vitamin B9 using enzyme linked immunosorbent assay technique. Results: The study showed that the level of vitamin B12 and folate B9 in hypothyroidism patients significantly lower compared to control group (p value < 0.001). Conclusion: Patients with hypothyroidism had noticeably low levels of vitamin B12 and folate B9, indicating that this may be involved in the etiology of hypothyroidism.