Background and Aim Non-syndromic hearing loss is a genetically heterogeneous disorder. Mutation in the GJB2 gene is a major cause of non-syndromic hearing loss in numerous countries. This study aimed to evaluate GJB2 mutations in 31 individuals with non-syndromic hearing loss Methods & Materials In this descriptive cross-sectional study, the required blood samples were collected from 31 individuals with non-syndromic hearing loss in Rasht and Bandar Anzali Cities, Gilan Province, Iran. After DNA isolation, the GJB2 gene was amplified by the PCR method and underwent sequencing. Ethical Considerations This study was approved by the Ethics Committee of the Islamic Azad University, Mashhad Branch (Code: IR. IAU. MSHD. REC. 1398. 027). Results In this study, 3 mutations were determined in 18 individuals with hearing loss. Accordingly, 35delG mutation had the highest frequency (48. 38%) in individuals with hearing loss as homozygote (n=14) and heterozygote (n=2). A patient with heterozygosity in V153I mutation and a patient with compound heterozygosity in 35delG/G200R mutation was determined. Conclusion It appears that 35delG mutation is a common mutation in the GJB2 gene in individuals with non-syndromic hearing loss in Guilan Province.