Objective:To explore the molecular genetic characterization of two families with aminoglycoside-induced and nonsyndromic sensorineural hearing loss. Methods:Blood samples were obtained from 7 maternal members and 1 married-in spouse of the two families. Genomic DNA was extracted with conventional method. Firstly, 9 hot spots for mutations in four most common pathologic genes, GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA, were screened with the DNA microarray to detect the deafness-associated mutations. The whole mitochondrial genomes and nuclear modifier genes TRMU and MTO1 of two probands were then PCR amplified and submitted for sequence analysis. Results:Mitochondrial 12S rRNA C1494T mutation was detected in all 7 maternal members of the two families. Sequence analysis of the complete mitochondrial genomes in two probands revealed the distinct sets of mtDNA polymorphism (52 other nucleotide changes), in addition to the identical 12S rRNA C1494T mutation. None of these 52 variants, however, were shown to be pathogenic. The whole mitochondrial genome of proband from each of the two families was established that they belong to mitochondrial haplogroups D4 and D5a respectively. No mutations were identified in either TRMU gene or MTO1 gene. Conclusion:C1494T mutation in the mitochondrial 12S rRNA gene is the main molecular mechanism responsible for the hearing loss in the two pedigrees, and the use of aminoglycoside antibiotics may enhance the phenotypic manifestation of deafness-associated mitochondrial mutation. Mitochondrial haplogroups and nuclear genes (TRMU and MTO1), however, seems not play a role in the phenotypic expression of C1494T mutation in these two families.