We aim to screen the mutations of 3 hearing loss (HL) genes (GJB2, SLC26A4, and 12S rRNA) in 71 cases with nonsyndromic hearing loss (NSHL) using microarray and SNPscan, and identify the roles of nonhotspot mutation of these genes in the screening of NSHL. Seventy-one cases with moderate or severe neurosensory deafness confirmed in our department from July 2014 to December 2015 including 25 Uyghur minorities and 46 Han Chinese were included in this study. The type of mutations in GJB2, SLC26A4, and 12S rRNA genes were detected using microarray and SNPscan, respectively. Statistical difference was noticed in the detection rate of the HL genes in 71 cases. Using microassay, deafness genes were identified in 10 subjects (14.08%), while 22 cases (30.98%) were confirmed with the presence of deafness genes using the SNPscan. Compared with the microarray, remarkable difference was noticed in the detection rate of SNPscan (P < .05). Nonhotspot mutation in GJB2, SLC26A4, and 12S rRNA genes played a crucial role in the pathogenesis of NSHL. SNPscan contributed to elevation of detection rate of NSHL in clinical practice.