The aim of this study was to assess the frequencies of three hemochromatosis gene (HFE) mutations in ethnic Roma/Gypsies in Slovakia. A cohort of 367 individuals representing general population and not preselected for health status was genotyped by TaqMan real-time PCR assay for C282Y, H63D and S65C mutations in HFE gene. A unique genetic profile was revealed: C282Y is found in the highest frequency of all Central European countries (4.90%), while the frequency of H63D mutation (4.09%) is lower than any reported in Europe so far. S65C mutation was not present in the cohort. These mutation frequencies can be explained rather by gene influx and genetic isolation than by genetic inheritance from a former Roma/Gypsy homeland.