Background: Polyomaviruses are small, circular, non-enveloped viruses with a diameter of about 40–50 nm, and have a double-stranded DNA molecule descended from the Polyomaviridae. 32 PyV strains are described, more than 10 of which are reported to infect humans. The current study aims to identify the sequence of the complete genome of the KI polyomavirus strain to study phylogenetic analysis and determine variation in the KI Polyomavirus sequence. Materials and Methods: One purified viral DNA sample of positive cases of KI Polyomavirus was sent for next-generation sequencing to identify whole genome sequencing. Results: The KI Polyomavirus sequence has been registered in the NCBI GenBank under the accession number LC776086.1 KI Polyomavirus. The current study showed that the KI Polyomavirus sequence has a length of 5040 bp. By comparing the sequences, it was found that there were many mutations, some of which were silent, and the other mutations caused a change in the amino acid. Ten reference strains from NCBI were used to create a phylogenetic tree for the studied clinical sample. Conclusion: The present results revealed that the studied strain LC776086.1 KI Polyomavirus showed great similarity with one sequence, EF127907 KI polyomavirus Stockholm 350, complete genome.