GJB2 gene encodes for a protein called "Gap junction β 2", responsible for the formation of membrane bound channels called "gap junctions" that in turn permit the transport of ions, and signalling molecules across neighbouring cells. The mutated form of this gene is majorly found to be responsible for Non-Syndromic Congenital deafness. Blood samples of the congenital deafness patients (n=25) were collected from Government Vellore Medical College Hospital, Vellore for analysis. With informed consent family and medical history were recorded. The molecular studies included DNA Extraction, Restriction Fragment Length Polymorphism (PCRRFLP) to infer mutation, in GJB2 gene. Single Nucleotide Polymorphism (SNP) in the natural variants of GJB2 protein using Bioinformatics tools was carried out. The various simulations that were run using bioinformatics tools helped us in categorizing Congenital Non Syndromic Deafness into Autosomal Dominant and Autosomal Recessive. The main aim of this study was to assess the type of mutation and the factors associated with the occurrence of Non Syndromic Congenital Deafness in Vellore population. This study would eventually help to deduce the pattern of inheritance following clinical features and the type of mutations present in the affected individuals. © Research India Publications.