Over the last few years, substantial progress has been made in developing strategies for the detection and characterization of various mutations causing beta-thalassemia. The Indian population comprises of numerous endogamous caste groups and beta-thalassemia is seen in almost all of them. Knowledge of the spectrum of beta-thalassemia mutations in the population is a prerequisite for successful implementation of a prevention programme. Among the different approaches available today, Denaturing Gradient Gel Electrophoresis (DGGE) offers a valid technical approach which is applicable for screening of known mutants and polymorphisms as well as in locating regions of DNA bearing unknown mutations. We analysed 356 unrelated beta-thalassemia heterozygotes by DGGE and detected 30 anomalous DGGE patterns. Fifteen mutations were characterized after sequencing 25 anomalous patterns. Of these, codon 10(GCC --> GCA) is a recently reported novel beta-thalassemia mutation while -28(A --> G) and codon 121(G --> T) are being reported for the first time in the Indian population. HbS and HbE also showed two anomalous DGGE patterns each. Framework (FW) linkage studies showed that four mutations were associated with different beta-globin gene frameworks. Linkage of IVSI-5(G --> C) and cap site +1(A --> C) to FW2 and 619-bp deletion to FW1 is being observed for the first time. Multiple DGGE patterns corresponding to the same mutation is one of the major drawbacks of this technique. In spite of this, if sufficient preliminary work has been carried out to compile a comprehensive catalogue of DGGE patterns; this is a powerful approach to characterize the mutation or to localize a small region of DNA in the case of rarer mutations.