Background: The avian influenza (AI) H5N1 viruses have caused nearly 600 fatal cases in the world since 2003. The H5N1 AI virus is still circulating in wild birds and domestic. One of the known of AI virus pathogenicity is the ability of the nonstructural protein (NS1 and NS2) to induce cytokine storm. Mutations of the NS1 and NS2 proteins may aggravate infections.Methods: Genetic sequences of NS gene that encodes the C - terminus domain of AI H5N1 virus were obtained by sequencing method using specific primer to amplify the NS gene. The primers design were done by using several bioinformatics software available then retested to determine the primer success in amplifying the target sequence. RT- PCR testing performed on four H5N1 AI virus isolates, followed by sequencing to determine the nucleotide sequences generated from the primer were prepared in this study. Results: Primers, which can be designed to amplify four chicken H5N1 isolates tested were two isolates from Bali (A/Ck/Bali-KH/2009 and A/Ck/Bali-T/2009), and two isolates from East Java (A/Ck/East Java-S/2009 and A/Ck/East Java-D/2009). Conclusion: The designated primer can be used in PCR amplification and sequencing of genes that express NS section C terminus of the protein NS1 and NS2 end portion. The primer design of H5N1 NS gene. (Health Science Indones 2014;2:83-8)