Multiple mutations in several ion channel genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, and KCNJ2) have been shown to cause autosomal dominant long QT syndrome (LQTS), a familial cardiac disorder that causes syncope, seizures, and sudden death. Due to their multiple loci and considerable size, mutation detection in these genes represents a challenge that is only partially met by the conventional screening method of single-stranded conformational polymorphism (SSCP). The recently introduced denaturing high-performance liquid chromatography (dHPLC) offers a promising new method for a fast and sensitive analysis of PCR-amplified DNA fragments. To test the applicability of dHPLC in the molecular diagnosis of LQTS, we first assessed a cohort of 192 patients from our International LQTS Registry for 14 previously identified mutations (including 10 different missense mutations, 1-bp, 2-bp, 3-bp, and 9-bp deletion mutations), and 2 polymorphisms in the LQTS potassium and sodium channel genes. Applying empirically determined exon-specific melting profiles, all mutations (including four previously undetectable by SSCP) were readily identified by dHPLC. We conclude that the dHPLC technology is a highly sensitive and efficient method for the molecular analysis of LQTS, and the same PCR amplicons developed for SSCP testing can be directly used for dHPLC assay.