In the present study, 24 H5N1 viruses were selected on the basis of the phylogenetic analysis of the HA gene sequences to study the genetic evolution of the NS1 gene of such viruses. The viruses were isolated from chickens and ducks in both live bird markets and backyard populations. NS1 gene was amplified, sequenced and the phylogenetic analysis of the obtained sequences revealed deletion of 5 amino acids at positions 80 to 84 in the NS1 protein along all Egyptian isolates under study. Ala149, which is a pathogeneicity marker for interferon antagonism was found in the studied indicating capability of these viruses to transmit between different avian species. PL motif ESEV was found in 6 isolates from chicken and ducks circulating in such population whereas 17 viruses have ESKV similar to those reported in human in 2006. Based on NS1 sequence analysis of the H5N1 Egyptian viruses, neither geographic nor species signature were reported. This study indicates independent evolution of NS1 gene in Egyptian viruses that is not related to HA gene grouping.