Background: Lumpy skin disease virus (LSDV) is an infectious viral disease which is an important serious skin disease of cattle. LSDV was isolated from skin biopsies collected from cattle which are clinically infected at Kaluobia governorate. Objectives: This study was designed for isolation and identification of LSD virus on CAM and MDBK cell culture and identification of isolated virus by FAT and PCR. Methods: A total 23 skin biopsies were used for virus isolation on chorio-allantoic membrane (CAM) of embryonated chicken eggs SPF at 9-11 day which showed pock lesions and CPE on MDBK cell culture. Isolated virus was identified by indirect fluorescent antibody technique (IFAT) using specific hyper immune serum against Lumpy Skin Disease Virus. Further identifications were carried out by polymerase chain reaction (PCR). Results: 15 samples were +ve for LSDV by conventional PCR.The results showed that 11/23 biopsies were positive by IFAT. Molecular identification of LSD virus by using RT-PCR, revealed positive for amplification of bands at the predicted molecular size (1926bp). Neutralizing antibodies against LSDV were (55) out of total 100 serum samples by serum neutralization test. Conclusion: Selection and processing of clinical specimens, viral isolation and PCR assay applied, for LSDV are much sensitive and rapid diagnostic tool of LSD reflecting their importance in controlling the rapid spread of disease in Egypt.