Detection of causal mutations is required for genetic counseling. Molecular modeling combined with patients' phenotype provides significant insight into structure-function relationship of factor (F)VIII molecule. Our objective was to identify defects in the gene of FVIII by a sensitive and simple scanning technique with high throughput in order to study molecular mechanisms by which novel amino acid substitutions may lead to hemophilia A. A cohort of 81 families with mild, moderate and severe hemophilia A negative in intron 22 inversion was studied. For detection of mutations in the FVIII gene a conformation sensitive gel electrophoresis (CSGE) was modified by multiplexing. Thirteen novel amino acid substitutions were studied by molecular modeling and a correlation with the cross-reactive material (CRM) phenotype was performed. In 74 families, 59 different mutations were detected. Six different mutations were recurrent in 21 unrelated families. Thirty-four novel mutations included 19 point mutations, four small insertions, nine small deletions and two complex mutations. Thirteen novel amino acid substitutions occurred at residues conserved in FVIII orthologs. Five of them were associated with a discrepancy between FVIII activity and antigen; another five with CRM reduced phenotype and one with undetectable FVIII antigen. Multiplexing of the CSGE significantly increased its throughput without substantial loss of sensitivity. Molecular modeling suggested mechanisms by which substitutions at residues 382 and 569, located outside the proposed FIXa-binding region, may influence FVIII/FIXa interaction. His2155 was predicted to participate in FVIII/VFW binding.