Abstract Background An inversion of intron 22 in the Factor VIII gene (Inv22) is the causative mutation for 45% of severe hemophilia A cases. Available methods for molecular diagnosis of Inv22 are generally tedious and not ideal for routine clinical use. Methods We report here a new method using a single closed-tube nested quantitative PCR (CN–qPCR) for rapid detection of Inv22. This method combines a 12-cycle long-distance PCR (LD–PCR) amplifying the int22h regions, followed by a duplex qPCR targeting two specific regions close to the int22h regions. All reagents were added to a single PCR mixture for the closed-tube assay. Sequential LD–PCR and qPCR was achieved by designing primers at substantially different melting temperatures and optimizing PCR conditions. Results Seventy-nine male hemophilia A patients of different disease severity were tested by both the CN–qPCR assay and the standard LD–PCR assay. CN–qPCR successfully made calls for all samples, whereas LD–PCR failed in eight samples. For the 71 samples where both methods made calls, the concordance was 100%. Inv22 was detected in 17 out of the 79 samples. Additionally, CN–qPCR achieved clear separation for 10 female carriers and 10 non-Inv22 females, suggesting the assay may also be useful for molecular diagnosis of female carriers. Conclusions This new CN–qPCR method may provide a convenient and accurate F8 Inv22 test suitable for clinical use.