The MLH1 gene responsible for colon cancer has been examined to identify functional consequences of single-nucleotide polymorphisms (SNPs). 16 SNPs have been identified in the MLH1 gene in which all are found to be nonsynonymous. Non synymous SNPs are relevant in many of the human inherited diseases since they change the aminoacid sequence of the protein. 56% of the identified nsSNPs have been reported as damaging. In the analysis of SNPs using SIFT, UTRscan, FastSNP and PolyPhen-2, it was recognized that rs41295284 and rs35001569 were responsible for the alteration in levels of expression. It has been concluded that among all SNPs of MLH1 gene, the mutation in rs41295284 and rs35001569 have the most significant effect on functional variation. Key words: Single nucleotide polymorphism, non-synonymous, colon cancer.