Chromosomal DNA prepared from 90 unrelated individuals, mainly of Caucasian origin, was screened for restriction fragment length polymorphisms in the 3' 220 kilobase pairs (kb) of the human thyroglobulin (Tg) gene. The probes used were Tg cDNA fragments and subcloned single-copy genomic segments, isolated from a human cosmid library. All in all, 1164 nucleotides were screened using 15 different restriction enzymes. The average number of nucleotides screened was 354 per individual. Only one polymorphism was found in these 1164 nucleotides, with a minor allele frequency of 2.2%. This polymorphism, which is located in an intervening sequence, was found in healthy individuals and in a family with hereditary congenital hypothyroidism due to a defect in the synthesis and structure of thyroglobulin. The Mendelian segregation of polymorphism and goiter in ten family members suggests that the rare variant is linked to a normal Tg allele and provides strong evidence for autosomal dominant inheritance of this Tg synthesis defect.