During routine avian influenza surveillance in Pakistan, a low-pathogenic avian influenza virus (LPAI) subtype H3N1 was isolated for the first time from domestic chickens. The higher seroprevalence of H3N1 was recorded in both commercial and domestic poultry in ecological zones of Pakistan where the geographical proximity with neighboring countries and attractive birding sites provide better opportunities for frequent movements of wild and migratory birds, and their intermingling with the local domestic and commercial poultry. Subsequent whole genome sequencing of this virus revealed a new introduction of a reassortant Eurasian avian strain, which was distinguishable from corresponding human and swine strains isolated elsewhere. Phylogenetically, the HA gene was mostly clustered with Nordic (Scandinavian) strains of influenza viruses, whereas the NA and PB1 genes showed a maximum nucleotide sequence homology with the Indian H11N1, and the PB2 gene was found to be closely related to the Altai H5N2. The Matrix and NP genes of H3N1 mostly clustered with the European avian influenza viruses (AIV), whereas its NS and PA genes showed maximum nucleotide homologies with the African (Egypt) AIV strains. A sequence and amino acid analysis revealed an LP motif, avian-like receptor specificity, potential glycosylation sites, and sensitivities to oseltamivir, zanamivir, and amantadine. Some point mutations possessed by this Pakistani AIV H3N1 were also found in human, equine, and swine H3 influenza viruses. This H3N1 isolate showed less nucleotide sequence homology with the previously known Pakistani AIV as compared with other Eurasian AIV strains.