Background: Guanine deletion 35delG in GJB2 exon 2 is the pathogenic mutation responsible for up to 70% of cases of congenital non-syndromic sensorineural hearing loss (NSHL) among Europeans. The early molecular diagnostic of hearing loss nature has become important while considering the cochlear implants. The purpose of this study was to establish the frequency of 35delG deletion in GJB2 gene among patients with severe NSHL and its prevalence among Moldovan residents with normal hearing. Material and methods: 40 patients with congenital bilateral profound NSHL and 300 individuals with normal hearing were examined for deletion 35delG, by using Custom TaqMan SNP genotyping Assay. Results: 12 (30%) patients with homozygous genotype for 35delG mutation were identified, whereas 8 patients (20%) were heterozygous. The study reported 4 (1.33%) carriers of 35delG mutation among 300 Moldovan individuals with normal hearing. Conclusions: The present study results suggest a need for including the 35delG molecular testing into the national program of neonatal screening of hearing loss. Considerations on the genetic carrier testing should be made in genetic counseling and family planning.