To determine the origin of sickle cell mutation in different ethnic groups living in southern Iran, we studied the haplotype background of the β^S and β^A genes in subjects from the provinces of Fars, Khuzestan, Bushehr, Hormozgan, and Kerman and from the islands of Khark and Qeshm. β-globin gene cluster haplotypes were determined using the PCR-RFLP technique. Detection of −α^3.7 deletion and β-thalassemia mutations were defined by PCR and reverse dot blot techniques, respectively. The framework of the β-globin gene was determined using denaturing gradient gel electrophoresis. We found that the β^S mutation in southern Iran is associated with multiple mutational events. Most of the patients were from two ethnic groups: Farsi speakers (presumably Persian in origin) from Fars province and patients of Arab origin from Khuzestan province. In both ethnic groups the Arab-Indian haplotype was the most prevalent. The frequencies of the Arab-Indian and African haplotypes in sickle cell anemia patients from the provinces of Fars and Khuzestan were similar. Among β^A chromosomes the Bantu A2 haplotype was the most prevalent. The decrease in α-globin production in SS patients and AS individuals appeared to be related to the reduction in mean cell volume and mean cell hemoglobin. The Arab-Indian haplotype gene flow into this region of Iran can be traced to the Sassanian Empire. It is likely that the influx of β^S genes linked to the Benin and Bantu haplotypes, of African origin, must have occurred during the Arab slave trade.