Hb D-Punjab [β121(GH4)Glu→Gln] is prevalent in the northern states of the Indian subcontinent. Due to inadequate data from Asian countries, the origin and spread of the Hb D-Punjab mutation are uncertain. In a study of sickle cell hemoglobinopathies, we detected the Hb D-Punjab mutation in 25 subjects from 11 unrelated Agharia families. Twelve cases were Hb S [β6(A3)Glu→Val]/Hb D-Punjab compound heterozygotes and 13 were Hb D trait carriers. In 76.0% of the cases, the β(D) gene was linked to haplotype I, whereas 24.0% had a novel haplotype. None of the haplotypes matched the β(A) haplotype of the local population. In view of the ancestral origin of the subjects and the high prevalence of the β(D) gene in the states of northern India, we suggest a North Indian origin for the β(D) mutation in our population. The finding of a novel haplotype in eastern India supports the hypothesis of a multicentric origin of this mutation.