Genetic testing for congenital or early-onset hearing loss patients has become a common diagnostic option in many countries. On the other hand, there are few late-onset hearing loss patients receiving genetic testing, as late-onset hearing loss is believed to be a complex disorder and the diagnostic rate for genetic testing in late-onset patients is lower than that for the congenital cases. To date, the etiology of late-onset hearing loss is largely unknown. In the present study, we recruited 48 unrelated Japanese patients with late-onset bilateral sensorineural hearing loss, and performed genetic analysis of 63 known deafness gene using massively parallel DNA sequencing. As a result, we identified 25 possibly causative variants in 29 patients (60.4%). The present results clearly indicated that various genes are involved in late-onset hearing loss and a significant portion of cases of late-onset hearing loss is due to genetic causes. In addition, we identified two interesting cases for whom we could expand the phenotypic description. One case with a novel MYO7A variant showed a milder phenotype with progressive hearing loss and late-onset retinitis pigmentosa. The other case presented with Stickler syndrome with a mild phenotype caused by a homozygous frameshift COL9A3 variant. In conclusion, comprehensive genetic testing for late-onset hearing loss patients is necessary to obtain accurate diagnosis and to provide more appropriate treatment for these patients.