With around 7,500 β-thalassemia (β-thal) births in India annually, preventive measures such as pre marital counseling and prenatal diagnosis are needed to decrease the incidence of the disease in the country. The predominant mutant alleles vary from the Middle East through India to Sri Lanka, as well as in different regions within a state in India. In the present study, involving the characterization of mutations in the Gwalior-Chambal region of Central India, IVS-I-5 (G>C) was found to be the most common allele followed by Hb E [β26(B8)Glu→Lys, GAG>AAG]/β-thal accounting for 10.83% and 619 bp deletion for 7.5% of the alleles. The high frequency of Hb E/β-thal in this region of Central India, unlike earlier reports from studies carried out in the state of Madhya Pradesh (Central India), mandates a more focused region-wise identification of common mutant alleles rather than the state as a whole.