During the Avian Influenza (AI) outbreaks in different areas of Pakistan (2003 - 06), a number of Avian Influenza Virus (AIV) isolates were recovered from the clinical samples. The samples were subjected to comparative diagnostic evaluation using in-ovo propagation, Virus Neutralization Test (VNT), rapid detection kits and Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR). The data revealed that RT-PCR technique was most sensitive and specific for the detection of Avian Influenza Virus subtypes and for differentially diagnosing it from other avian respiratory pathogenic viruses. These isolates were further utilized for the development of multiplex RT-PCR. A multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) was developed and standardized for the detection of type A influenza viruses, Avian Influenza Virus (AIV) subtype H7, H9 and H5 haemagglutinin gene with simultaneous detection of 3 other poultry respiratory pathogens Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious laryngotracheitis virus (ILTV). Seven sets of specific oligonucleotide primers were used in this study for the M-Gene of AIV and haemagglutinin gene of subtypes of H7, H9 and H5 of AIV. Three sets of other specific oligonucleotide primers were used for the detection of avian respiratory pathogens other than AIV. The mRT-PCR DNA products were visualized by Agarose Gel Electrophoresis and consisted of DNA fragments of 1023bp for M-Gene of AIV, 149bp for IBV, 320bp for NDV and 647bp for ILTV. The second set of primers used for m-RT-PCR of H7N3, H9N2 and H5N1 provided DNA products of 300bp for H7, 456bp for H5 and 808bp for H9. The mRT-PCR products for the third format consisted of DNA fragments of 149bp for IBV, 320bp for NDV, 647bp for ILTV, 300bp for H7, 456bp for H5, 808bp for H9. The sensitivity and specificity of mRT-PCR was determined and the test was found to be sensitive and specific for the detection of AIV and other poultry respiratory pathogens. In the present study, multiplex PCR technique has been developed to simultaneously detect and differentiate three most important subtypes of AIV’s alongwith 3 most common avian respiratory pathogens prevalent in poultry in Pakistan. The non-structural 1 (NS1) protein of avian influenza viruses has been earlier described as a remarkably conserved protein amongst type A influenza viruses, however with xxivsubsequent findings of is truncation during extensive circulation in poultry has led to further investigate its mutation in association with point mutations simultaneously occurring in more variable genes such as HA and NA. Apart from affecting any of the biological functions of these viruses, these mutations may affect the immunogenic component(s) of these viruses, affecting the efficacy of prevalent vaccines. To establish if Pakistani H7N3 Avian influenza viruses undergo any truncation in non-structural genes, the non-structural gene 1 (NS1) of 22 H7N3 Avian influenza A viruses isolated from commercial and domestic poultry was sequenced and compared phylogenetically. The isolates included in the present study were both of low pathogenecity (LPAI) and highly pathogenic strains (HPAI) of H7N3 avian influenza viruses as observed in the field with regards to their mortality rates. These isolates circulated in N.W.F.P, Punjab, and Sindh areas of Pakistan from 1995 to 2005. Size variation in the predicted amino acid sequence of each NS1 was revealed with two different levels of carboxy-terminal truncation in those isolates. Of the 22 isolates analyzed, 02 isolates A/Chicken/Pakistan/NARC-100/04 and A/Chicken/Pakistan/NARC-1282/04 encoded a full length NS1 protein of 230 amino acids, whereas 20 encoded a truncated protein of 217 amino acids. The isolates exhibiting the truncated carboxy terminal NS1 protein, clustered together and appeared to be closest to A/Duck/Jiang Xi/6146/03 (H5N3), A/Duck/Hong Kong/610/79 (H9N2) and A/Aquatic Bird/Korea/CN-1/04 (H3N6) at the nucleotide level and amino acid level. In contrast, the nucleotide sequence of one of the isolates with the full length NS1 protein (A/Chicken/Pakistan/NARC-1282/04) showed 99.9% nucleotide homology and 99.6% homology to a set of Italian H7N3 isolates of Turkey from 2002 at the NS1 gene e.g A/turkey/Italy/8912/2002(H7N3) and A/turkey/Italy/214845/02(H7N3). The other isolate (A/Chicken/Pakistan/NARC-100/04) with the full length NS1 protein showed the highest homology (96%) with the NS1 gene of an H5N7 subtype virus A/mallard/Denmark/64650/03. Out of these 22 H7N3 isolates sequenced for the NS1 gene, 6 isolates from the Northern Parts of Pakistan were further sequenced for the HA and NA genes. One of the isolates had an untruncated NS1 whereas 5 were truncated. The 5 H7N3 isolates with truncated NS1 sequenced were HPAI, for the HA gene and showed the presence of typical highly pathogenic pattern of deduced amino acid sequence at the HA cleavage site. The xxvphylogenetic analysis of these H7N3 isolates indicated a close resemblance to other Pakistani isolate sequences in the GenBank, with the next closest resemblance to the H7N3 isolate from a Peregrine Falcon in U.A.E in the GenBank besides the other Pakistani isolates. The untruncated isolate for the NS1 gene, A/Chicken/Pakistan/NARC- 1282/04, showed a typical low pathogenicity cleavage site sequence at the HA cleavage site. Phylogenetic Analysis of this isolate indicated a close resemblance to Italian H7N3 isolates especially A/Chicken/Italy/682/2003 (H7N3) and A/turkey/Italy/8535/2002 (H7N3). The NA gene was analyzed for the presence or absence of a stalk region in the isolates sequenced. The 5 truncated H7N3 isolates for the NS1 Gene and HP for HA gene had a stalked NA protein as in H7N3 isolates reported in wild birds showing a close resemblance to other previously sequenced H7N3 Pakistani isolate sequences in the GenBank, whereas the untruncated NS1 H7N3 isolate also showing a LPAI cleavage site sequence A/Chicken/Pakistan/NARC-1282/04 had a deleted NA stalk region, deduced amino acid sequence showing a deletion of 24 amino acids in concordance with other Italian H7N3 isolates reflecting a probable introduction of a highly circulating virus in domestic poultry. It was concluded from the present study that the H7N3 isolates from Pakistan show slow antigenic drift and continue to evolve in a slow manner during a ten year period in the poultry population. With information obtained from the data on NS1, HA1 and NA, continuous monitoring of circulating viruses is possible and subsequent production of homologous vaccines from field strains is key to the control of HPAI in poultry.