Hemophilia B is an X-linked recessively inherited bleeding disorder afflicting humans across all socio-economic as well as racial groups. A wide range of mutations showing high heterogeneity has been reported in different populations. Thus, it has been difficult to adopt a cost-effective strategy for the genetic diagnosis of hemophilia B families. We report the presence of a common G10430A mutation in exon d of the factor IX gene, wherein the highly conserved Gly 60 residue of the first epidermal growth like domain was changed to Ser in 22 out of 22 moderately severe to mild hemophilia B patients originating from Gujarat. None of the eight Gujarati severe hemophilia B patients, 30 normal Gujarati men, and 20 moderately severe to mild hemophilia B patients belonging to other communities showed the presence of this mutation. This mutation occurred in the same haplotype background thereby suggesting a 'founder effect.' The direct detection of this G10430A mutation can be used for accurate carrier detection and prenatal diagnosis in mild to moderate factor-IX-deficient patients belonging to the Gujarat state of western India.