Aim: Mutations in mitochondrial tRNA (mt-tRNA) genes are associated with various diseasesincluding colorectal cancer. The relationship between changes in tRNA genes and cancer is increasingly receiving attention. The focus of this study is to comprehend the possible relationship between mttRNA mutations and colorectal cancer incidence and progression. Study design: The present study we sequenced mitochondrial DNA of 25tumour+25 adjacent normal] samples belonging to Colorectal cancer tissues by Next generation sequencing [NGS] technology. The identified tRNA variants were screened for pathogenicity and structural changes that lead to hampering the function of mt-tRNA protein synthesis. Results: We have identified 10 mt-tRNA mutations across tRNAIle G4264A, tRNAIle A4281G, tRNAAla T5594A, tRNASer[AGC] T12261C, tRNALeu[CUN] G12300C, tRNALeu[CUN] A12306C and tRNALeu[CUN] T12329A. Apart from SNP’s, three deletions were identified which are potentially pathogenic: tRNASer[AGC]A12212del, tRNASer[AGC] T12261del, tRNALeu[CUN] A12328del. Of the identified mutations five of them were novel and were likely pathogenic and reside in extremely conserved positions. Further analysis of copy number suggests the presence of low copy numbers when compared to the matched normal tissues [p<0.05]. Hence, it's conceivable these mutations hinder mitochondrial functions, contributing to colorectal carcinogenesis. Conclusion: Collectively, our data suggest tRNA genes are common sites for pathogenic mutations linked to colorectal cancer, potentially serving as biomarkers for screening and diagnosis.