Beta-thalassemia is a common inherited disease, resulting from one or more of a total of more than 200 different mutations in the beta-globin gene (HBB). Efficient and reliable mutation-screening methods are essential in order to establish appropriate prevention programs for at-risk populations based upon a molecular diagnosis. We have developed a rapid and highly-specific mutation screening test for the diagnosis of beta-thalassemia by coupling heteroduplex and primer-extension analysis based on the denaturing high performance liquid chromatography (DHPLC) system. A total of 161 healthy heterozygous Taiwanese carriers featuring 10 different HBB mutations and 30 patients exhibiting 12 different compound heterozygous or homozygous HBB mutations were subjected to DHPLC. The elution profile for the heteroduplex analysis of DHPLC could be successfully used to identify the common disease-causing mutations of HBB. To further confirm the sequence variants, we developed a technique combining multiplex primer-extension analysis coupled with DHPLC for the genotyping of eight common disease-causing mutations in the HBB gene. Overall, by coupling heteroduplex and primer-extension analysis based upon DHPLC, we were able to unambiguously identify the most-common beta-thalassemia mutations corresponding to more than 99% of HBB alleles among the Taiwanese population. In conclusion, compared to classic approaches to mutation screening for this malady, we suggest that DHPLC is an excellent technique to be applied to the genetic screening of prenatal and postnatal individuals as a part of a diagnosis program for beta-thalassemia and provides a more-efficient, economic, and sensitive means to undertake such a screening program.