Infectious bronchitis virus (IBV) is incriminated in a variety of clinical conditions in poultry. IBV has a potential to mutate under field conditions and due to this multiple serotypes and variants of the IB virus circulates in commercial and backyard poultry. In the present study seroprevalence levels of different known serotypes of IBV such as, Mass-41, 4/91, D274, D1466, IT-02, D388 & D8880 were first determined in non IB-vaccinated poultry from different provinces of Pakistan during 2012 to 2015. The data showed high seroprevalence of multiple serotypes of IBV indicating high level of diversity in the circulating serotypes/variants of IBV in this country. In addition to this, a post-IBV vaccination base line was developed by sero-monitoring among healthy poultry in response to IBV vaccines, providing a permanent reference for future monitoring of post-vaccination antibody titers For further IBV investigations, different diagnostic techniques to be used in this study were optimized in this study. Moreover, studies regarding the determination of tissue tropism of one of the new IBV isolates along with evaluation of its co-infection potential with Avian Influenza virus H9N2 and ORT were also carried out. For the detection and typing of locally circulating Pak-IBV isolates following techniques were first optimized including, haemagglutination (HA), haemagglutination inhibition (HI), Indirect-Enzyme linked immunosorbent assay (ELISA), restriction fragment length polymorphism (RFLP), reverse transcriptase polymerase chain reaction (RT-PCR), real time reverse transcriptase polymerase chain reaction (QRT-PCR), virus neutralization (VN). Using these techniques, a total of 3187 clinical samples were processed for IBV detection, out of this 871 were IBV positive. Moreover, subtype detection revealed that 45.2% was Mass-41, 51.3% was IBV serotype 793-B and 3.4% were variants or un-identified. Furthermore, 871 RT-PCR positive samples were propagated upon in-ovo inoculation in specific pathogen free (SPF) embryonated chicken eggs. Out of this 55 IBV isolates were subjected to RFLP analysis that grouped the isolates into three segments, first was designated as 4/91 like group, second was designated as Mass-41 like group and the third was designated as IBV variant. The sequence and phylogenetic analysis of three IBV isolates from each RFLP group revealed that the first isolate, Pak IBV-786, shared sequence homology of the range of 25 99.1%-99.5% with 4/91 like strains from China, India, Russia, Morocco, Japan and Iran or GI-13 IBV lineage. The second isolate, Pak IBV-1113 shared 98% sequence of its sequence with IBV vaccine strains of Ma5 and M41 from Brazil, India, USA, Egypt, China, Iran, Thailand and Poland. The third and new Pak isolate, IBV-973, shared 91-93% of its sequence with the Indian strains of IBV earlier reported in India only, of GI-24 lineage. This strain of IBV variant has been first time reported from commercial poultry in Pakistan and its subsequent molecular characterization revealed that this virus is in fact a new serotype of IBV earlier only reported from India. Pathogenesis and co-infection studies on the isolate Pak-973 further highlighted biological characteristics of the new Pak-variant, which led us to believe that this variant may be contributing significantly towards the development of super complex of Respiratory Tract Infection, with or without the involvement of AIV H9N2. The sequence and phylogenetic analysis of different Pak IBV isolates reflected here that the genome of IBV is under a continuous process of evolution, due to point mutations, selective pressure (vaccine) and recombination events. So as like other RNA viruses, the IBV control is most likely to succeed upon using serotype specific vaccines (Homologous vaccines), as carried out elsewhere. It would, therefore be highly appropriate to recommend the incorporation of strain Pak-973 in commercial poultry vaccines being used in this country. INDEX WORDS: Infectious bronchitis virus, Pak-variants, Spike glycoprotein, RFLP, Real time RT-PCR, Co-infection, Tissue tropism