Lumpy skin disease virus (LSDV) was isolated, from naturally infected cattle that have a history of previous vaccination with live attenuated sheep pox virus (SPV) vaccine. The virus was isolated on chorio-allantoic membrane (CAM) of specific pathogen free (SPF) embryonated chicken eggs (ECE) and identified by agar gel precipitation test (AGPT) and neutralization test using specific hyperimmune serum against LSDV and SPV. Characteristic intracytoplasmic inclusion bodies was detected in trypsenized cell of infected CAM stained with H&E. Laboratory studies for characterization of isolated LSDV revealed that it was stable at a wide range of pH, but it was inactivated by exposure to 56 0C for 15 minutes. Treatment of isolated LSDV with lipid solvents (20% ethyle ether and chloroform) reduced the virus titer 3.2 and 4.4 log respectively after 24 hrs at 4 0C .On cross neutralization test
complete neutralization of isolated LSDV was obtained with both reference LSDV and SPV antisera. Cattle vaccinated with live attenuated SPV vaccine under experimental condition found to be protected against natural field infection with LSDV.