Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE ): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation. HFE-related hereditary hemochromatosis (HH) is an autosomal disorder of iron metabolism causing iron overload status and affecting Caucasian populations with a prevalence of approximately 1 in 300 (1). Clinical consequences of iron overload include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, and endocrine pancreatic disease, which can be easily prevented by early diagnosis and iron depletion by phlebotomy. Three recurrent missense mutations in the HFE gene have been associated with HH: C282Y, a cysteineto-tyrosine amino acid substitution caused by an 845 G>A transition at codon 282 within exon 4; H63D, a 187 C>G transversion within exon 2 that results in a change at codon 63 from histidine to aspartate, and S65C, a serineto-cysteine substitution generated by a 193 A>T transversion in exon 2. C282Y has been well characterized as