Summary. Background: Inversions of F8-intron 22 (Inv22) and F8-intron 1 (Inv1) are responsible for 45–50% of severe hemophilia A cases. Objective: In order to improve the molecular diagnosis of Inv22 and Inv1, and to enable rapid discrimination of Inv22-type 1 and Inv22-type 2 patterns, int22h-mediated deletions (Del22) and duplications (Dup22), we developed a genotyping system based on a novel inverse shifting-polymerase chain reaction (IS-PCR) approach. Methods: IS-PCR involved BclI restriction, followed by self-ligation to create ‘BclI circles’, and finally PCR analysis. Three PCR analysis tests were developed: (i) Inv22-diagnostic for a pattern-sensitive detection of deleterious mutations (Inv22 and Del22) from non-deleterious variants (Dup22 and normal); (ii) Inv1-diagnostic; and (iii) Inv22-complementary for discrimination between Inv22 and Del22, and between Dup22 and normal. For rapid molecular analysis of F8, the Inv22 and Inv1 diagnostic tests can be performed simultaneously. The optional Inv22-complementary test need only be used for specific purposes. Results and Conclusions: Diagnostic tests were validated using previously studied samples. IS-PCR evaluated carrier mosaicisms and performed robustly over wide ranges of DNA qualities and procedural conditions. IS-PCR improved the molecular diagnosis of hemophilia A. This genotyping strategy may potentially be adapted to virtually all known rearrangements in the human genome.