BackgroundThe majority of non-syndromic hearing loss (NSHL) patients result from causative mutations in GJB2, SLC26A4 and mitochondrial 12S rRNA genes. Accurate detection of these genetic mutations is increasingly recognized for its clinical significance to reduce incidence and guide individual treatment of NSHL. Current methods for clinical practice are labor intensive, expensive or of low sensitivity. MethodsGenomic DNA from 7 newborns not passing the hearing screening and 94 newborns passing the hearing screening were analyzed for the common mutations using high resolution melting analysis (HRMA) and Sanger sequencing. ResultsOur newly developed HRMA allowed the hot-spot mutations of GJB2 c.176_191del16 and c.235delC, SLC26A4 IVS7-2A>G and mitochondrial 12S rRNA 1494C>T and 1555A>G to be detected by melting profiles based on small amplicons. HRMA can distinguish different content mutant DNA from wildtype DNA, with a detection limit of 5%. Moreover, the results were highly concordant between HRMA and Sanger sequencing. ConclusionsThese results indicate that HRMA could be used as a routine clinical method for prenatal diagnosis and newborn genetic screening due to its accuracy, sensitivity, and rapid, low-cost and less laborious workflows.