Objective:To determine the prevalence and characteristics of deafness-causing mutations in Connexin26(Cx26,GJB2)gene and mitochondrial DNA A1555G with non- syndromic hearing impairment(NSHI). Methods:The peripheral blood samples were obtained from the students of Nanjing Deaf-Blind School and healthy people in Jiangsu province. Using polymerase chain reaction (PCR),the code region of mitochondrial DNA and GJB2 gene were amplified. The GJB2 235delC mutation and mtDNA A1555G mutation was distinguished by restricted enzyme digestion. All of the subjects were sequenced. Results:Fifteen kinds of polymorphism about GJB2 gene,were found in 162 cases of controls and 9 kinds of pathologic mutations of GJB2 gene(V27I,E114G,I203T,V37I,176-191del16,235delC,299-300delAT,T123N and 504insGCAA) were identified in 135 patients. GJB2 235delC is the main pathogenic mutation site in GJB2 gene,which was found in 37cases(18 cases were homozygosis,19 cases were heterozygosis). The mitochondrial DNA A1555G mutation was not found in those subjects. Conclusion:The mutation of Cx26 genes is much responsible for non-syndromic hearing impairment in this region. Our data support the view that specific combinations of GJB2 mutation exist in different populations. 235delC is the main pathogenic mutation site in GJB2 gene in Chinese. The incidence of mtDNA 12SrRNA A1555G mutation in the deaf population of Nanjing,Jiangsu Province is lower than the average level of the general Chinese deaf population.