Dengue fever virus (DENV) poses a significant public health risk in tropical and subtropical regions across the world. Although the dengue fever virus (DENV) exhibits significant genetic diversity and has the potential to evolve, there is a lack of comprehensive research on the comparative genomics and evolutionary dynamics of the virus in Pakistan. Phylogenetic analysis demonstrated the circulation of all four dengue virus serotypes (DENV-1, - 2, - 3, and - 4) with prevalent genotypes III and V within DENV-1, cosmopolitan genotype within DENV-2, genotype III within DENV-3, and genotype I within DENV-4 during 2006-2014. Based on the complete envelope region, genome-wide residue signature and genetic diversity indicate that there is a high level of genetic diversity among DENV-1 strains, while DENV-3 strains exhibit the least genetic diversity. Comparative analysis of all four DENV serotypes revealed that certain codons in DENV-2 and -4 were subject to strong purifying selection, while a few codon sites in the envelope region showed evidence of positive selection. These findings provided valuable insights into the comparative genomics and evolutionary pattern of DENV strains reported from Pakistan. Whether those characteristics conferred a fitness advantage to DENV-1 genotypes within a specific geography and time interval warrants further investigations. The findings of the current study will contribute to tracking disease dynamics, understanding virus transmission and evolution, and formulating effective disease control strategies.
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