Many studies determined the demographic and ethnic border of patients with beta (β)-thalassemia mutations and their migration. The effective way to health care policy of β-thalassemia is to prevent homozygote births and reduce the severity of the disease. The objectives of this study contributed to investigating the molecular and serologic characteristics of β-thalassemia patients in Iraq. Peripheral blood samples were collected from 97 β-thalassemia patients and 32 healthy control subjects. Quantitative sandwich enzyme-linked immunosorbent assay was performed to measure serum ferritin, 25-hydroxy vitamin D, and 8-hydroxydeoxyguanosine (8-OHdG) levels. Further, the β-globin mutation detection assay involving an extensive screening of β-globin mutations by direct Sanger DNA sequencing and gap-PCR was performed to detect the Δ619 deletion mutation. The results revealed that compared with the control subjects, the β-thalassemia patients showed significantly decreased vitamin D levels and significantly increased serum ferritin and 8-OHdG levels (all, P<0.001). Molecular analysis detected 9 types of mutations in the β-thalassemia patients, only 2 of which, namely IVS II-1 G>A and IVS 1-5 G>C, have been previously reported in Iraqi studies, whereas the remaining 7, namely IVS-II-666 C>T, CD2 CAT>CAC, IVS-II-850 G>A, IVS-II-16 G<C, Hb King's Mill, Hb Saveh, and IVS-II-81 C>T, have never been reported in the Iraqi population. This study showed that the serum ferritin and 8-OHdG levels were significantly higher, and the serum 25-hydroxy vitamin D levels were significantly lower in the β-thalassemia patients than in the control subjects. Moreover, the results revealed seven newly identified mutations among Iraqi β-thalassemia patients and 2 previously reported mutations.