Heteroduplex analysis of polymerase chain reaction (PCR)-amplified factor IX (FIX) sequences in eight hemophilia B pedigrees localized the causative hemophilia mutation to a single exon in each case. Subsequent PCR-based direct DNA sequence analysis identified two novel FIX mutations and six recurrent mutations. Three of the eight pedigrees represent sporadic hemophilia B, and direct mutation analysis facilitated hemophilia carrier diagnosis in each case.