Molecular characterization of hemophilia B at gene level has become an indispensable tool for a proper genetic counseling in carriers and for a closer surveillance of inhibitor development in several severe forms. Our study was aimed at characterizing the molecular defects in the factor IX (FIX) gene in hemophilia B families in Aragon, a center-east region of Spain. Direct sequencing of all regions of likely functional significance of the FIX gene was performed in the screened 18 hemophilia B families. Quantitative techniques, such as multiplex ligation-dependent prove amplification reaction, were carried out only in patients in whom no mutation was found. We have identified the molecular events responsible for hemophilia B in 16 unrelated families (eight with mild hemophilia B and eight with severe hemophilia B). Out of all families studied, we have found 14 missense mutations and two nonsense mutations; still we were unsuccessful in determining the genetic defects in two severe and unrelated families. Of the 16 characterized mutations, 14 of them lie in the protease domain in which one mutation, A233T, was surprisingly found in three unrelated families. We also report and discuss the pathogenicity of F314L, a novel mutation found in the protease domain. Our molecular data reflect a notable heterogeneity of the mutational spectrum mainly in the protease domain of FIX. This is the first mutation report on the disease in Aragon, Spain.