Hereditary hemochromatosis is an inherited disorder of iron metabolism, characterized by high absorption of iron by the gastrointestinal tract leading to a toxic accumulation of iron in various organs and impaired organ function. Three variants in the HFE gene (p.C282Y, p.H63D, and p.S65C) are commonly associated with the development of the disease. Of these, p.C282Y homozygotes are at the highest risk. Compound heterozygotes of p.C282Y along with p.H63D or p.S65C have reduced penetrance. Furthermore, p.H63D homozygotes are not at an increased risk and little is known about the risk associated with homozygocity for p.S65C. Our current clinical assay for the three common HFE variants utilizes the LightCycler platform and paired probes employing fluorescent resonance energy transfer. To increase throughput and decrease costs, we developed a method whereby automated extraction was combined with unlabeled probes and differential melt profiles to detect these variants using the LightCycler 480 instrument. Using this approach, 43 samples extracted with three different extraction platforms were correctly genotyped. These data demonstrate that the newly developed assay to genotype the HFE mutations p.C282Y, p.H63D, and p.S65C, combined with high-throughput extraction platforms, is accurate and reproducible and represents an alternative to previously described tests.