Target exon resequencing using Massively Parallel DNA Sequencing (MPS) is a new powerful strategy to discover causative genes in rare Mendelian disorders such as deafness. We attempted to identify genomic variations responsible for deafness by massive sequencing of the exons of 112 target candidate genes. By the analysis of 216randomly selected Japanese deafness patients (120 early-onset and 96 late-detected), who had already been evaluated for common genes/mutations by Invader assay and of which 48 had already been diagnosed, we efficiently identified causative mutations and/or mutation candidates in 57 genes. Approximately 86.6% (187/216) of the patients had at least one mutation. Of the 187 patients, in 69 the etiology of the hearing loss was completely explained. To determine which genes have the greatest impact on deafness etiology, the number of mutations was counted, showing that those in GJB2 were exceptionally higher, followed by mutations in SLC26A4, USH2A, GPR98, MYO15A, COL4A5 and CDH23. The present data suggested that targeted exon sequencing of selected genes using the MPS technology followed by the appropriate filtering algorithm will be able to identify rare responsible genes including new candidate genes for individual patients with deafness, and improve molecular diagnosis. In addition, using a large number of patients, the present study clarified the molecular epidemiology of deafness in Japanese. GJB2 is the most prevalent causative gene, and the major (commonly found) gene mutations cause 30-40% of deafness while the remainder of hearing loss is the result of various rare genes/mutations that have been difficult to diagnose by the conventional one-by-one approach. In conclusion, target exon resequencing using MPS technology is a suitable method to discover common and rare causative genes for a highly heterogeneous monogenic disease like hearing loss.