Telomeres are specialized structures at the end of eukaryotic chromosomes that maintain genomic stability by preventing chromosomal rearrangements and thereby enabling semi-conservative replication of telomeric DNA. The length of telomeric DNA is retained by telomerase that balances between the processes that lengthen and shorten the telomeres. In human chromosomes, six telomere-associated proteins namely- TRF1, TRF2, POT1, RAP1, TIN2, and TPP1 form the shelterin complex, that is essential for maintenance of telomeric integrity. The human POT1 and TPP1 play a major role in protecting the ssDNA overhangs, formed due to the end replication problem. These proteins along with other repair complexes protect the telomere overhangs from cellular repair complexes. However, shelterin functionality can become compromised due to mutations in any of the six genes and can lead to unwarranted repair of the human telomeres. In cancer and transformed cells, telomerase activation replenishes the telomeres while also, recruiting repair proteins at the telomeres. With an aim to evaluate the functional consequence of non-synonymous single nucleotide polymorphisms (nsSNPs) in POT1 gene variants, and resulting changes that affect its interactions with TPP1, this research was carried out using computational tools. The overall outcomes revealed 16 POT1 gene mutations that were likely to impact the protein function. Of these 9 mutations, viz., P357S, H437P, V439G, P475L, G534C, P537S, F566C, M587T, and C591W showed that the altered POT1 function impacted its interaction with TPP1 protein. The binding affinity of POT1 with ssDNA overhangs was also changed. A wet-lab follow-up study using site-directed mutagenesis and yeast hybridization techniques can help exploit underlying mechanisms affecting stable association of these two shelterin components. © 2020