High coagulation factor VIII (FVIII) levels are a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in two Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before the age of 50, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defect. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1 and a large part of intron 1), which co-segregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a ≥2-fold up-regulation of the F8 mRNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In conclusion, we report the first thrombophilic defect in the F8 gene (designated "FVIII Padua") associated with markedly elevated FVIII levels and severe thrombophilia in two Italian families.