Discovery of deafness genes has progressed but clinical application lags because of the genetic heterogeneity. To establish clinical application strategy, we reviewed the frequency and spectrum of mutations found in Japanese hearing loss patients and compared them to those in populations of European ancestry. Screening revealed that in Japanese, mutations in GJB2, SLC26A4, and CDH23, and the mitochondrial 12S rRNA are the major causes of hearing loss. Also, mutations in KCNQ4, TECTA, COCH, WFS1, CRYM, COL9A3, and KIAA1199 were found in independent autosomal dominant families. Interestingly, spectrums of GJB2, SLC26A4, and CDH23 mutations in Japanese were quite different from those in Europeans. Simultaneous screening of multiple deafness mutations based on the mutation spectrum of a corresponding population using an Invader panel revealed that approximately 30% of subjects could be diagnosed. This assay will enable us to detect deafness mutations in an efficient and practical manner in the clinical platform. We conclude that specific racial populations may have unique deafness gene epidemiologies; therefore, ethnic background should be considered when genetic testing is performed. Simultaneous examination of multiple mutations based on a population's spectrum may be appropriate and effective for detecting deafness genes, facilitating precise clinical diagnosis, appropriate counseling, and proper management.