Autosomal recessive non-syndromic hearing loss (ARNSHL) a most frequent hereditary type of hearing impairment, exhibit tremendous genetic heterogeneity. We aimed to determine the contribution of three common DFNB loci (DFNB4, DFNB28, and DFNB93), and mutation analysis of Gap Junction Beta-2 gene (GJB2) and GJB3 genes in ARNSHL subjects in southern Iran.Thirty-six large ARNSHL pedigrees (167 individuals) with at least two affected subjects (72 patients) were included in this descriptive study from Hormozgan Province of Iran, during 2014 - 2015. The variation of GJB2 and GJB3 genes were screened using direct sequencing method. The negative samples for GJB2 and GJB3 genes mutations were analyzed for the linkage to DFNB4, DFNB28, and DFNB93 loci by genotyping the corresponding short tandem repeat (STR) markers using polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) methods.DNA sequencing of GJB2 were identified heterozygous mutation (964 C/T) in 13.88% of the studied families. Three missense mutations (788G/A, 284C/T and 973G/C) were also detected in coding region of the GJB3 gene. The 284C/T mutation in the GJB3 occurs in compound heterozygosity along with the 964T/C mutation in the GJB2 in one family. Finally, we found no evidence of linkage to either of DFNB4, DFNB93 and DFNB28 loci.Highlighting the hypothesis that a genetic interaction between GJB2 and GJB3 genes could be lead to ARNSHL, however, no evidence of linkage to the DFNB loci was found. 284C/T variant in GJB3 gene might be pathogenic when accompanied by variant in GJB2 in a digenic pattern. However, further large-scale familial and functional studies are required to challenge this hypothesis.