The aim of the present study was to examine the pathogenesis and tissue tropism of the Avian Influenza Virus (AIV) H9N2 and Newcastle disease virus (NDV) in the broiler birds in single and mixed infections. The endemics of virulent strains of Avian Avulavirus-1s (AAvV-1s) and low-pathogenic H9N2 avian influenza viruses (AIV) are being continuously reported in Pakistan. The repeated outbreaks are the main source of high economic losses to the poultry industry. In this study, genetic characterization and pathotyping of five AAvV-1s and two H9N2 viruses have been investigated from the NDV and H9N2 suspected samples collected during 2013-14. It was observed through phylogenetic analysis that all the NDV isolates were related to sub genotype VIIi with high similarity of 97.9% to 99.8% with similar viruses in this clade. The gene sequences of haemagglutinin (HA) of two AIV were analyzed and phylogenetic analysis reveals genetically closely-related resemblance to H9N2 viruses classified into Mideast group-B and sub-lineage B2. The two strains were classified as LPAIV in poultry on the basis of sequence of amino acids at proteolytic cleavage site of haemagglutinin gene with PAKSSR/G. Our findings highlight the potential risk of ND and AI in poultry and continued active surveillance is needed to monitor the transmission of these viruses. To study the pathogenesis and tissue tropism in the broiler birds, 210 day old broiler chicks were divided in six groups of 35 each. Five groups of broiler birds were challenged with single NDV, single H9N2 and their mixed-infections. Sixth group was kept as a disease free control group with no challenge of the virus. Ten birds were slaughtered on 3rd, 5th and 7th day post infection (dpi). The organs of digestive, respiratory, lymphoid, circulatory, urinary and nervous systems were collected for histopathology and immunohistochemical examination. Microscopically, different lesions were observed in different organs in single ND and H9N2 infected and co-infected groups. IHC was used to detect the NDV and H9N2 in same organs. NDV and H9N2 were detected separately at different locations of above mentioned organs in groups A and B. Both viruses were detected simultaneously in co-infected groups C, D and E. No virus was detected in group F. It is concluded that histopathological lesions were more severe in the organs of birds of group A (infected with virulent NDV), relatively less lesions were observed in co-infected groups (C, D and E) as compared to birds of group A and lesions were milder in group (B) infected with LPAIV (H9N2). The efficacy of the commonly used commercial vaccines for Newcastle disease (ND) and low path avian influenza (LPAI) H9N2 were evaluated against field virus in broiler chicks. One hundred one-day-old commercial broiler chicks were divided into four groups (A to D) with an equal number of birds per group. Group A and B were vaccinated against H9N2 and NDV, respectively, at day 7 of age while group C served as positive infected control for H9N2 and group D for NDV. Serum samples from birds in all groups were tested for presence of antibodies against H9N2 and NDV at day 21 of age. Subsequently, on day 28 of age, groups A and C were challenged with the field strain of H9N2 virus, and Group B and D with NDV. Birds were monitored for a period of 2 weeks for development of any clinical signs and mortality. The geometric mean titer were high in groups A (4.90) and B (7.3), and low in the unvaccinated groups C (0.7) and D (1.1). The highest and lowest value of H9N2 antibody titer detected through ELISA were 1.498 and 0.502, respectively. The S/P ratios greater than 0.5 were considered positive. The highest and lowest value for NDV antibody titer detected through ELISA were 783 and 882, respectively. Serum samples with titer greater than 396 were considered positive and indicated vaccination or other exposure to NDV. On histological examination severe congestion, necrosis, degeneration, hemorrhages and leukocyte infiltration were observed in intestine, lungs, trachea and bursa of Fabricius of the non-vaccinated group post-infection. Mild tissues changes were observed in the vaccinated group. It can be concluded from the findings that the commonly used commercial vaccines may provide effective protection against the circulating H9N2 and ND virus in broiler birds by producing protective antibody titer.