Part of the molecular basis of congenital hypothyroidism (CH) has been elucidated by the identification of molecular defects in pituitary- and thyroid-specific genes in patients with various subtypes of hypothyroidism. So far identified genetic defects only explain a small proportion of cases of hypothyroidism. Thus novel research strategies are required to isolate more tissue-specific genes involved in the pathogenesis of CH at present considered ‘idiopathic’ from a molecular perspective. We applied serial analysis of gene expression to human thyroid tissue and developed a computational substraction method to identify tissue-specific genes. The result has been the identification of three genes preferentially expressed in the thyroid gland. The first one encodes part of the thyroid oxidase (THOX2) system. We linked mutations in the THOX2 gene with idiopathic cases of transient and permanent CH. The second transcript identified, DEHAL1, encodes the protein responsible for the recycling of iodine in the thyroid gland and represents the candidate gene for a specific subtype of CH. The third one encodes NM41, a protein currently under investigation which shows features characteristic of the cystine-knot family of proteins, typically involved in early development.