Objective: To determine the prevalence and characteristics of mitochondrial DNA (mtDNA) 12S rRNA gene and tRNASer (UCN) gene mutations in Chinese subjects with non-syndromic hearing impairment. Methods: The peripheral blood samples were obtained from 135 patients with non-syndromic hearing loss and 126 normal hearing controls. Genomic DNA was isolated from the peripheral leukocytes of all participants using the Puregene DNA Isolation Kits. The subject’s DNA fragments spanning the entire mitochondrial 12S rRNA gene and tRNASer(UCN) gene were PCR amplified. Each fragment was purified and subsequently analyzed by direct sequencing to identify deafness-associated mutations. Results: There were totally eleven 12S rRNA sequence variants detected in 135 hearing-impaired subjects. Of those,4 variants are known deafness-associated mutations with variable frequencies of 4.4% in A827G,1.5% in T961C,0.7% in T1095C and 1.5% in A1555G. Other variants,such as T1005C,C1048T,T1119C,G709A,C752T and A1382C,seem to be polymorphisms rather than causes of disease. On the other hand,we did not find C1494T mutation in the 12S rRNA gene and any of the known deafness-associated mutations in tRNASer (UCN) gene in all individuals. Conclusion: The 12S rRNA gene may be a hot spot for mitochondrial mutations causing non-syndromic hearing loss in the Han nationality population in Nanjing,Jiangsu province,with a total carrier frequency of 8.1% for deafness-related mutations.