Sensorineural hearing loss (HL) is one of the most frequent clinical features in patients with mitochondrial diseases caused by mitochondrial DNA (mtDNA) mutations, and hearing is impaired in over half of all cases with mitochondrial disorders. This study analyzed 373 patients with suspected hereditary HL using an extensive and rapid suspension-array screening system for 29 major mtDNA mutations, including the m.1555A>G homoplasmic mutation in the MT-RNR1 gene, which causes non-syndromic sensorineural HL and aminoglycoside-induced HL, and the m.3243A>G heteroplasmic mutation in the MT-TL1 gene. This method is rapid and suitable for large-scale screening because universal 96-well plates are available for use, and because an analysis of each plate can be completed within 1 h. This system detected five different mtDNA mutations in 24 of the 373 (6.4%) patients. The m.1555A>G and m.3243A>G mutations were detected in 11 (2.9%) and 9 (2.7%) patients, respectively. In addition, three mutations, that is, m.8348A>G in the MT-TK gene, m.11778G>A in the MT-ND4 gene and 15498G>A in the MT-CYB gene were detected in one patient for each. This screening system is useful for the genetic diagnosis and epidemiological study of both syndromic and non-syndromic HL.