Summary. β‐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 β‐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 β‐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 β‐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 β‐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 β‐thalassaemia, and strongly suggests that this technique can be used successfully in other genetic diseases.