Objective mitochondrial DNA (mtDNA) mutation are one of the important causes of deafiness.In particular, the 12S rRNA gene is the hot spots for mutations associated with both aminoglycoside ototoxicity and nondyndromic deafiness.Methods in this report, a total of 318 Chinese pediatric hearing-impaired subjects were recruited from otology clinics in the Zhejiang Province, China.Results these subjects underwent clinical, genetic evaluation and molecular analysis of 12S rRNA.Mutational analysis identified 34 variants in the 12S rRNA gene in this cohort.The incidences of the known deafness-associated 1555A>G, 1494C>T and 1095T>C mutations were 9.1%, 0.6% and 1.25% in this cohort, respectively.Other mtDNA variants were evaluated by structural and phylogenetic analysis.Of these, the 839A>G and 1452T>C variants could confer increased sensitivity to aminoglycosides or nonsyndromic deafness as they were not present in 449 Chinese controls and localized at highly conserved nucleotides of the 12S rRNA.1 However, other variants appeared to be polymorphisms.Conclusion these data further support the idea that mitochondrial 12S rRNA is one of major targets for aminoglycoside ototoxicity.These data have been providing valuable information to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside antibiotic therapy, and eventually to decrease the incidence of deafness.