A total of four clinical cases consisting of abortion, repeat breeding and rhinitis were investigated for IBR. The MDBK cell lines were used for virus isolation. A total of two isolates (IBR-1 and IBR-2) were isolated from the clinical cases. Both the isolates showed characteristic CPE on MDBK cell line consisting of rounding, grape like bunch appearance and cell detachment. Intranuclear inclusion bodies were observed in the stained MDBK infected cell preparations. The isolates were further confirmed by serum neutralization test. Molecular analysis of two Infectious bovine rhinotracheitis (IBR) virus isolates was done on the basis of study of band pattern of cleaved fragment of their DNA after digestion with restriction enzyme. The extracted DNA of IBR isolates (IBR-1 and IBR-2) when subjected to electrophoretic mobility on 0.5 per cent agarose gel showed single compact band of intact DNA. When the isolate IBR-1 subjected to Bam HI restriction enzyme digestion showed six bands, which represent A+B, C+D, E, F, G and H+I fragment whereas IBR-2 did not show any band. The isolation and identification of IBR on MDBK cell line along with molecular analysis was found to be highly specific and reliable technique for the confirmation of the diagnosis.