ß-thalassemia is the most prevalent single-gene disorder. Since no viable forms of treatment are available, the best course is prevention through prenatal diagnosis. In the present study, the prevalence of ß-thalassemia was extensively investigated in the South Indian population, especially from the state of Andhra Pradesh. Screening for causal mutations was carried out on genomic DNA isolated from patient blood samples by using the routine reverse dot blot (RDB) and amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) techniques. DNA sequencing was performed wherever necessary. Among the nine mutations identified, four, including IVS-1-5 (G-C) (IVS1+5G>T), codon 41/42 (-TTCT) (c.124_127delTTCT), codon 15 (G-A) (c.47G>A), and HbS (sickle mutation) (c.20A>T) mutations, accounted for about 98% of the total positive cases. Two mutations viz. codon 8/9 (+G) (c.27_28insG) and HbE (codon 26 G-A) (c.79G>A) exhibited a very low frequency of occurrence, whereas the IVS-1-1 (G-T) (IVS1+1G>T) and the 619 bp deletion (c.366_494del) mutations were absent. We also identified certain rare mutations during the diagnostic evaluation. Gene sequencing confirmed the codon 30 (G-C) (c.92G>C) mutation and the rare codon 5 (-CT) (c.17_18delCT) and IVS-II-837 (T-G) (IVSII-14T>G) mutations. This is the first report of the IVS II 837 mutation in the Indian population. We also report a novel diagnostic application during RDB-based screening for the detection of the (c.92G>C) mutations. Such a comprehensive mutation screening is essential for prenatal diagnosis of ß-thalassemia and control of this highly prevalent monogenic disorder in the Indian population.