Background & aim: Deafness (HL) is the most common sensorineural disorder. The frequency of hearing impairment in the world is one in every 500 newborns. However, in Iran, due to the high rate of consanguineous marriages, this amount is estimated to be two to three times higher. So far, more than 120 genes causing non-syndromic deafness (NSHL) have been known in the world. Among these, GJB2 gene mutations are the most common cause of autosomal recessive NSHL. The aim of this research was to determine and investigate the frequency and spectrum of GJB2 gene mutations in the non-syndromic deaf population of Semnan province. Methods: The present practical study was conducted in 2021 on 50 patients with non-syndromic hearing loss from 50 unrelated families. First, the very common 35delG mutation was screened by allele-specific polymerase chain reaction (ASPCR). Then, in order to identify other mutations of the GJB2 gene, patients who were negative or heterozygous for the 35delG mutation were analyzed by trench sequencing for all exonic regions and splicing sites of the GJB2 gene. The collected data were analyzed using descriptive statistical tests. Results: In the present study, 100 chromosomes (50 patients) were examined. 12 chromosomes (12%) indicated mutation in GJB2 gene. Among these, the share of each of the identified mutations 35delG, IVS1+1G>A and c. 94C>T: p. Arg32Cys in the deaf population of Semnan province, respectively,7, 4 and 1 percent. Thus, the common 35delG mutation includes 58. 34% of all identified mutations. Also, unlike other populations studied in Iran, the IVS1+1G>A splice site mutation had a relatively high prevalence in the deaf population of Semnan province. Conclusion: The results of the present study indicated that GJB2 gene mutations comprised only 12% of mutations causing hearing loss in the deaf population of Semnan province, which emphasizes the importance of using next generation sequencing techniques to identify other genes causing hereditary hearing loss in this population. Such an approach will significantly aid in carrying out genetic counseling, prenatal diagnosis and clinical management of hearing loss in Semnan province.