Haemophilia A (HA) is an X-linked recessive bleeding disorder caused by a lack or decrease of coagulation factor VIII activity. The molecular diagnosis of HA is challenging and a variety of different mutations have been identified throughout the F8 gene. Our aim was to detect the causative mutation in 266 HA patients from Emilia-Romagna region (Italy) and in all suspected carriers. Molecular analysis of F8 in 201 HA patients (152 index cases) was performed with a combination of several indirect and direct molecular approaches, such as long distance polymerase chain reaction, multiplex ligation-dependent probe amplification, denaturing high performance liquid chromatography and direct sequencing. The analysis revealed 78 different mutations, 23 of which were novel, not having been reported in national or international databases. The detection rate was 100%, 86% and 89% in patients with severe, moderate and mild HA, respectively. The information provided by this registry will be helpful for monitoring the treatment of HA patients in Emilia-Romagna and also for reliable genetic counselling of affected families in the future.