In the past two decades, substantial advancements have occurred in identifying many different deafness genes. Along with the discovery of these deafness genes, our understanding of the biology of hearing has advanced significantly. Clinically, therapeutic intervention based on the molecular mechanism of hearing loss has become possible. Taking into account the onset of gene therapy in 2024, the molecular diagnosis of hearing loss is likely to become essential in the near future. However, the genetic diagnosis of hearing loss is difficult as many genes can cause similar hearing loss phenotypes, making it impossible to know which genes are involved without genetic testing. To find the responsible gene, the screening strategy has changed from one-by-one gene screening to the simultaneous analysis of multiple genes. Targeted exon resequencing of selected genes using next-generation sequencing (NGS) technology will enhance molecular diagnosis in the clinical setting and make it possible to identify rare gene pathogenic variants that cause deafness in individual patients. With the accumulation of information on pathogenic variants and the phenotypes of deafness-causing genes, it has become clear that deafness genes show a wider range of phenotypes than previously thought, encompassing both non-syndromic and syndromic hearing loss, as well as congenital and late-onset hearing loss. The most remarkable aspect of these advancements in terms of clinical applications is that ENT clinicians can now make highly accurate molecular diagnoses prior to choosing the appropriate therapy. This process enables a clearer understanding of the mechanisms involved, more appropriate and precise treatment selection, and greatly improved genetic counseling. Genetic testing has become crucial to the provision of personalized therapeutic interventions for individual patients.