OBJECTIVE: This study examined the mutation spectrum and frequency of three prevalent pathogenic genes in patients with non-syndromic hearing loss (NSHL) from Gansu Province, China. METHODS: We analyzed 452 NSHL patients from five special education schools across several cities in Gansu Province using SNPscan technology to determine the mutation spectrum of mtDNA 12S rRNA, GJB2, and SLC26A4 genes. RESULTS: Among the 452 patients evaluated, mutations in the GJB2 gene were observed in 91 cases (20.13 %), mutations in the SLC26A4 gene in 81 cases (17.92 %), and homoplasmic mutations in mtDNA 12S rRNA in 26 cases (5.75 %). Significant differences in GJB2 mutations were observed between Han patients and those of Hui, Tibetan and Mongolian ethnicity (χ CONCLUSION: Our findings demonstrate variations in the mutation spectra of the GJB2, SLC26A4, and mtDNA 12S rRNA genes across different ethnic groups, highlighting ethnic variations in mutation prevalence. This study expands the understanding of the genetic mutation spectrum associated with deafness in Gansu and supports the enhancement of molecular diagnostic accuracy for diverse ethnic populations in the region.