Beta-thalassaemia is a common inherited disorder of haemoglobin synthesis worldwide, with an estimated 3-10% frequency in certain regions. Rapid, accurate genotyping methodologies for specific, causative mutations of the beta-globin gene are needed for pre- and postnatal screening and diagnosis of this disease in different ethnic populations. In this study, we performed a novel multiplex primer extension (PE) reaction in combination with denaturing high-performance liquid chromatography (DHPLC) for simultaneously detecting and genotyping the five most common molecular lesions in the beta-globin gene [codons (CDs) 41-42 (-TCTT), IVS-2-654 (C-->T), - 28 (A-->G), CD17 (A-->T) and CD71-72 (+ A)] in Chinese populations. This method involved the amplification of beta-globin target sequence followed by a purification step, a multiplex PE reaction that did not require labelled oligonucleotides, and a fully-denaturing DHPLC analysis on the Transgenomic Wave DNA fragment analysis system. In a blinded study, this technique accurately genotyped 100% (120/120) of samples previously characterized by reverse-dot blot and direct sequencing, and was used successfully for prenatal diagnosis of beta-globin mutations in six Chinese families. This study validated the combined PE/DHPLC approach as simple, rapid, highly accurate and cost-effective for use in genotyping common disease-causing mutations, including substitutions, insertions and deletions in beta-thalassaemia, and strongly suggests that this technique can be used successfully in other genetic diseases.