Background Gene variants are responsible for more than half of hearing loss, particularly in nonsyndromic hearing loss (NSHL). The most common pathogenic variant in SLC26A4 gene found in East Asian populations is c.919-2A > G followed by c.2168A > G (p.H723R). This study was to evaluate their variant frequencies in patients with NSHL from special education schools in nine different areas of Southwest China's Yunnan. Methods We performed molecular characterization by PCR-products directly Sanger sequencing of the SLC26A4 c.919-2AG and c.2168 A > G variants in 1167 patients with NSHL including 533 Han Chinese and 634 ethnic minorities. Results The SLC26A4 c.919-2A > G variant was discovered in 8 patients with a homozygous state (0.69%) and twenty-five heterozygous (2.14%) in 1167 patients with NSHL. The total carrier rate of the c.919-2A > G variant was found in Han Chinese patients with 4.50% and ethnic minority patients with 1.42%. A significant difference existed between the two groups (P < 0.05). The c.919-2A > G allele variant frequency was ranged from 3.93% in Kunming to zero in Lincang and Nvjiang areas of Yunnan. We further detected the SLC26A4 c.2168 A > G variant in this cohort with one homozygotes (0.09%) and seven heterozygotes (0.60%), which was detected in Baoshan, Honghe, Licang and Pu`er areas. Between Han Chinese group (0.94%) and ethnic minority group (0.47%), there was no statistical significance (P > 0.05). Three Han Chinese patients (0.26%) carried compound heterozygosity for c.919-2A > G and c.2168 A > G. Conclusion These data suggest that the variants in both SLC26A4 c.919-2A > G and c.2168 A > G were relatively less frequencies in this cohort compared to the average levels in most regions of China, as well as significantly lower than that in Han-Chinese patients. These results broadened Chinese population genetic information resources and provided more detailed information for regional genetic counselling for Yunnan.