HFE-linked hereditary haemochromatosis is a common autosomal recessive disease among Caucasians. The primary pathogenetic mechanism is excessive absorption of iron, which is deposited in various organs with their subsequent damage. In 1996 the gene responsible for haemochromatosis was detected--the HFE gene and its major mutation C282Y. The discovery of further mutations followed. Two sites of point mutations in the HFE gene, C282Y and H63D, are associated with more than 80% of haemochromatosis cases. Another mutation-- S65C--was detected on 8% of chromosomes of haemochromatosis patients, which were negative for mutations C282Y or H63D. The objective of this study was to identify the allele frequency of S65C and other HFE mutations in the Czech population. DNA extracted from 481 randomly selected newborn screening cards (Guthrie cards) from all over the country was analysed by PCR-RFLP. No (0%) sample was identified as homozygous for S65C or C282Y mutation and 8 (1.67%) were homozygous for H63D mutation. Twelve (2.49%) samples were S65C heterozygous, 33 (6.86%) samples were C282Y heterozygous, and 128 (26.61%) were H63D heterozygous. Of these, 11 (2.29%) carried one copy of each mutation, i.e. were compound heterozygous. Two samples were S65C/H63D compound heterozygous and nine were C282Y/H63D compound heterozygous. Allele frequencies for S65C, C282Y, and H63D were 1.25% (95% CI, +/- 0.70), 3.43% (95% CI, +/- 1.15), and 14.97% (95% CI, +/- 2.25), respectively. The observed genotype frequency for S65C, C282Y, and H63D mutations in the Czech Republic agrees with those reported for other Central European populations.