β-Thalassemia (β-thal) is a common single gene autosomal recessive disorder resulting in severe anemia due to reduced or absent β-globin polypeptide synthesis. The disease is caused by mutations in the β-globin gene; eight common mutations are proposed to cause the majority of β-thal in India. However, the occurrence of a region-specific mutation spectrum in India has also been suggested. We had earlier carried out analyses of the β-globin gene mutation spectrum from southern Indian states of Andhra Pradesh and Karnataka. In the current study, we have analyzed three of 73 transfusion-dependent patients visiting a referral hospital in Karnataka State, South India, who did not carry any of the 22 common β-globin gene mutations as determined by reverse dot-blot analysis. The IVS-II-837 (T>G) (β(+)) (HBB:c.316-14TG) mutation was detected in two of the three patients analyzed suggesting a higher occurrence of the mutation in β-thal patients in Karnataka when compared to other regions of India. The rare polyadenylation (poly A) site (T>C) (AATAAA>AACAAA; β(+)) mutation was detected in the third patient. The IVS-II-837 mutation was also identified in asymptomatic carrier parents during routine high performance liquid chromatography (HPLC)-based Hb A(1c) screening in suspected diabetes patients. This is the first report of the identification of β-thal trait through HPLC-based diabetes screening in India, revealing the importance of linking diabetes screening with screening for thalassemia.