The ancient Silk Road (also called "Northwest Silk Road") in Northwest China, starting from Xi'an, passes through Gansu, Xinjiang, Central Asia, West Asia, and the land passage connecting the Mediterranean countries. The aim of the present study was to determine the frequency of mitochondrial DNA12SrRNA m.1555A>G mutation in a total of 2417 cases of nonsyndromic deaf-mute patients representative of the general population of Shaanxi, Gansu, Qinghai, Ningxia, and Xinjiang along the Silk Road. Enzyme digestion and direct sequencing were applied to identify sequence variations. The carrier frequency of mitochondrial DNA12S rRNA m.1555A>G mutation was estimated to be 5.21% (126/2417) in the studied population. In detail, the carrier frequency of Uighur and Hui was 1.62% (3/185) and 3.29% (10/304), respectively, compared with 6.09% (113/1856) that of Han. There was a statistically significant difference between Uighur and Han (chi-square test, chi(2) = 6.437, p = 0.011 and p < 0.05), whereas no significant difference in m.1555A>G mutation spectrum or prevalence of mitochondrial DNA12SrRNA was found between Uighur and Hui or Hui and Han. In the 126 m.1555A>G mutation carriers, 52 cases were found to have a clear history of using aminoglycoside antibiotics. Results suggested that the application of aminoglycoside antibiotics in this region is an important reason for higher incidence of m.1555A>G mutation in the deaf-mute population.