H9N2 is a low pathogenic avian influenza virus that causes respiratory tract infection in poultry industry worldwide. Viral neuraminidase (NA) plays a vital role in pathogenicity due to its sialidase activity. In this study, H9N2 virus from diseased birds was isolated at GP laboratory, Lahore. Then viral NA gene was amplified, sequenced and analyzed using different bioinformatics tools. Results revealed that high pathogenicity of the isolated strains is due to G127S and S450L mutations in comparison to low pathogenic avian influenza virus and they have evolved from A/partridge/Pakistan/260/2015(H9N2) virus. The evolution of the isolated chicken virus from partridge virus shows its phylogenetic relationship. High pathogenicity of our isolates is attributed to the amino acid substitutions: G127S, I192V, V307M and S450L in the isolates with accession numbers MH105293, MH105294 & MH105295. Though H9N2 in Pakistani poultry is regarded as a low pathogenic influenza virus but our current study shows it to be highly pathogenic with high mortality >75%. By using SWISS MODEL software, NA protein model was also constructed, which is more stable than wild type with 119kJ/mol& 145kJ/mol energy values respectively. Furthermore, positions at G127S, I192V, V307M, S450L, D141N, V254I and V312I have significant antigenic variability, as well as more beta sheets than alpha helices affecting the protein structure, function and stability. This study will help the poultry vaccine manufacturers to design the customize in silico vaccine or neuraminidase inhibitors to meet the challenges arose in local poultry industry of Pakistan due to H9N2 viral infection. (C) 2020 PVJ. All rights reserved