Lumpy skin disease (LSD) of cattle is a highly contagious viral skin disease causing sever economic losses. In Egypt, Protection of cattle against LSD was carried out using sheep poxvirus vaccines. In the present study, confirmative identification of previously isolated lumpy skin disease virus (LSDV) and commonly used sheep poxvirus (SPPV) vaccine (RM65 strain) nucleic acids was carried out by conventional polymerase chain reaction (PCR) and dot blot hybridization (DBH) depending on attachment protein gene (192 bp) of capripoxviruses. Differentiation between this LSDV isolate and SPPV vaccine (RM65 strain) was performed via PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) and real time-PCR melting curve analysis depending on 390 bp genome fragment in which EcoRV sites is present in case of SPPV and absent in LSDV. Therefore, the amplified 390 bp genome fragment was digested by EcoRV in case of SPPV and not digested in LSDV isolate. Also, real time-PCR melting curve analysis revealed that melting temperature (Tm) of LSDV isolate was 78°C where Tm of SPPV vaccine was 76 °C. from this study, we concluded that PCR-RFLP and real time-PCR melting curve analysis could be used to differentiate between cattle infected with LSDV and those vaccinated with SPPV vaccine.