Hereditary hemochromatosis is known as the most common genetic disorder among individuals of European genetic background. It is possible to find 2 mutations closely placed in the HFE gene (H63D and S65C) and this proximity can cause errors when genotyped by real-time polymerase chain reaction (PCR) genotyping assay. The aim of this study was to develop a hydrolysis probe-based PCR assay for detection of the H63D and S65C mutations without interference from on each other. Herein the study involved the standardization of an improvement of the real-time PCR 5′ nuclease assay to detect the desired mutations close placed using a same probe system. The assay analytical properties performances were tested, including the primers selectivity and detection limits. Also, the interexaminer reproducibility and repeatability of assay were estimated in 30 blood samples. Others 153 results of samples were compared with reference method (PCR_RFLP) and the accordance of the results evaluated by Fleiss’ κ method. The results of variation of interexaminer reproducibility and repeatability of assay were not statistically relevant (P<0.001). The comparison between the 2 methods by Fleiss’ κ analysis showed that 5′ nuclease assay identified the H63D and S65C haplotype as well as the reference method in all 153 tested samples. Our results showed that novel method probe-based real-time PCR were capable to detect 2 adjacent polymorphisms without errors in genotyping.