A large variety of mutations within the genes encoding hepcidin (HAMP) and hemojuvelin (HJV) have been identified in patients with the severe iron overload disorder juvenile hemochromatosis (JH). The aim of the present study was to evaluate the molecular background of JH in patients from central parts of Europe. Sequence analyses of HAMP and HJV were performed in seven JH patients from six families from Germany, Slovakia, and Croatia. For detection of the G320V mutation in HJV, a rapid polymerase chain reaction-based assay was developed. No mutations were found within the HAMP gene. Six of seven (86%) JH patients carried at least one copy of the G320V mutation within the HJV gene. Four of these patients were homozygous for the G320V mutation. In addition, two novel HJV mutations were identified (C119F and S328fsX337). Taken together, the present study demonstrates that molecular analysis of the HJV gene is a powerful tool for an early and reliable diagnosis of JH. As in affected patients from Greece, the G320V mutation seems to be widely distributed among JH patients from central parts of Europe. Therefore, detection of the G320V mutation could identify the majority of JH cases from these regions non-invasively.