Variations in mitochondrial genome have been found to be associated with hearing loss. Of these, the mitochondrial 12S rRNA and tRNASer(UCN) are the hot-spots for pathogenic variants associated with deafness. To understand the putative role of mitochondrial DNA (mtDNA) variants in hearing loss, we recently screened the variants in mitochondrial genomes in patients with deafness from the Hangzhou area of Zhejiang Province, People’s Republic of China (PRC). In this study, we describe a maternally-inherited Han Chinese family with high penetrance of hearing loss, notably, the penetrance of hearing loss in this family were 80.0 and 40.0%, when the aminoglycoside was included or excluded. Three matrilineal relatives in this pedigree exhibited different levels of hearing loss with different age at onset. In addition, sequence analysis of the complete mitochondrial genome showed the presence of the well-known C1494T pathogenic variant in the