Deafness or hearing loss is one of the most prominent genetic disorders in human beings. Hearing loss is caused by a number of environmental and genetic factors. The genetic factors involve about 130 genes which have role in hearing loss. Among them, the mutations in channel protein connexin genes GJB2, GJB6 and in mtDNA genes resulting in hearing losses. The GJB2 and GJB6 genes codes for connexin-26 and connexin-30 proteins, which help the potassium K+ ions recycling in the inner ear cells and activates/trigger the neurotransmitters.The neurotransmitters are signaling moleculeswhich here receive and transfer, the nerve impulses between the central nervous system and sense organs, recognizing sound accordingly. For unraveling the mechanism of Non-syndromic Hearing Loss (NSHL), a precise laboratory protocols was established and employed, for identification two nuclear genes i.e. exon2 of GJB2, the exon1 of GJB6 gene, and detection of mutations in three mitochondrial genes viz. MTRNR1, MTRNR2 and MT-TV. For elaborating the pattern of mutations in NSHL patients, 1500 oral swabs were collected from the deaf patients belonging to Abbottabad, Bannu, Charsadda, Haripur, Mansehra, Mardan, Peshawar, Swabi and Swat districts of Khyber Pakhtunkhwa Province (KP), Pakistan. We observed mutations in 5 genes i.e. 2 nuclear (GJB2 and GJB6) and 3 mitochondrial genes (MTRNR1, MTRNR2 and MT-TV) in 700 (47%) out of the total 1500 deaf patients. Whereas, the rest of deaf patients (800) might be having mutations in other deafness related genes. We observed higher incidence of deafness related gene mutations in males (68%) as compared to the females (32%). The mutations in GJB2 and GJB6 genes showed prevalence of 1.6 and 0.67%, respectively whereas, in mitochondrial genes i.e. MTRNR1, MTRNR2 and MT-TV, the mutation rate was 0.8, 0.73 and 0.53%, respectively. The protocol includes the isolation of total genomic DNA from the oral swab epithelial cells through modified phenol-chloroform method of DNA extraction. The DNA was amplified through thermo scientific polymerase chain reaction (PCR) and gene cleaned through manual washing of PCR product with 75% ethanol with step wise centrifugation. The sequencing was carried out in gene analyzer machine, through Sanger’s sequencing method. After sequencing of desired genes, all sequences were verified and confirmed by comparison with reference sequences at NCBI gene bank. We identified some known and many novel mutations in sampled deaf patients including indel, missense and nonsense mutations in targeted genes. The identified mutations in GJB2 gene include V27C, D46E, N54K, K61R, E110G, A78S, A78P, D66N, W77C, W77L, K15E, K103N, V153I, 120F, F115V, D46A, V38A, W24*, E119* c.327G>A, c.186C>T, c.228A>T, c.120A>G and c.240G>A, . The identified mutations in GJB6 gene were c.41delA, c.42delC, c.43delA, c.31delG, c.ins374-375(16nt), c.ins320321(19nt), p.K15Q, p.A88T, p.A92D and p.A149S mutations. The mutations in MTRNR1 gene were, 1349 T>G, 1420T>G, 1438A>G, 1440 G>A, 1442 G>A, 1492 A>C, 1544 A>T, 1545 G>A, 1546A>T, 1554G>A, 1575 T>G, 1577A>G and 1598 G>A variants in MTRNR1 gene. The mutations identified in MTRNR2 gene included, 1671 G>A, insT>1711, 1735 A>C, 1754 G>A, 1811 A>G, 1814 A>C, delT> 1872, 1888 G>A, 1899 G>A, insT>1960 and insG>1990.Similarly, the mutations identified in MT-TV gene included, 1604G>T, 1604G>A, 1606G>A, 1609T>G, 1610 A>C, 1625 A>C, 1641G>T, and 1644G>A. Analyses of the mutations data revealed that these mutations cause frame shift, missense and nonsense mutational changes in the gene expression and thereby result in hearing losses. It was further confirmed by protein alignment, that these mutations also changes the structural configurations of Cx26 and cx30 proteins, as well as affect the mitochondrial DNA dysfunction, which impair sound recognition mechanism. Our study provides reliable protocols for DNA extraction, gene cleaning and sequencing of concerned responsible genes for hearing loss and thereby screening deaf patients on one hand, and on the other hand we have established a baseline for gene mutations in deaf patients of Khyber Pakhtunkhwa. These findings can be used for genetic counselling, disease diagnostics and gene therapy etc.