The aim of this study was to analyze the role of HFE mutations in blood donors with iron parameters suggesting iron overload, taking into account the regional distribution of HFE mutations in Italy. We studied 5880 subjects undergoing evaluation for blood donation eligibility, from different areas of Italy. Abnormal iron parameters were defined as transferrin saturation (TS) >50% or >45% and serum ferritin (SF) >300 or >250 microg/ml in males and females, respectively. Subjects with increased TS and/or SF were re-tested and typed for HFE mutations C282Y and H63D. A total of 548 individuals had increased iron parameters at first testing. In total, 179/548 were available for retesting, and in 109 increased TS and/or SF were confirmed. Increased TS was confirmed in 25 individuals, among whom three were C282Y homozygotes and six were compound heterozygotes for C282Y and H63D. Increased TS was more frequent in northern Italy than in southern regions. In individuals with increased TS and/or SF, the frequency of C282Y and H63D was 0.13 and 0.21 in northern-Italy versus 0.05 and 0.45 in southern Italy (P=0.004 for H63D). Nine out of 10 individuals carrying hemochromatosis-associated genotypes (including compound heterozygosity for C282Y and H63D) originated from northern regions. Among controls, the allelic frequencies of C282Y and H63D were 0.037 and 0.16 in the northern regions and 0.015 and 0.16 in the southern regions. In conclusion, over one-third of individuals with persistently altered TS carried hemochromatosis-associated genotypes, confirming that a diagnostic approach based on TS and genotyping of selected cases may represent a viable screening procedure.