Background The genus Capripoxvirus (CaPV) within the family Poxviridae com-prises three closely related viruses, sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) causing sheep pox (SPP), goat pox (GTP) and lumpy skin disease (LSD) in small ru-minants and cattle respectively. LSD has emerged in Europe in 2015 and first incursions of SPP in the European Union were reported in Bulgaria and Greece in 2013. Live attenuated SPPV vaccines are widely used in many countries to control SPP and GTP. With the in-creasing number of reports on SPP in previously vaccinated sheep herds, it is imperative to develop new diagnostic tools for differen-tiation of SPPV field strains from attenuated vaccine strains. Objective This work aimed at identifying appropriate diagnostic targets to de-velop assays for the rapid and accurate differentiation SPPV vaccine strains from SPPV field isolates and other CaPVs. Methodology To identify a suitable molecular target for the development of these assays, the full genomes of several SPPV vaccines strains and SPPV field isolates were compared. A unique 84-base pair nucleotide dele-tion located between the DNA ligase and the B22R gene was exploit-ed to develop a gel-based PCR, and a region containing a 48bp de-letion within the B22R gene of SPPV vaccines strains only, as well as species-specific nucleotide difference between SPPV field isolates, GTPV and LSDV, was targeted to develop a HRM assay. Results The gel-based assay was readily able to differentiate SPPV vaccines from field isolates. However, this method alone could not differen-tiate SPPV field isolates from GTPV and LSDV. In contrast, the HRM based method allowed the differentiation of SPPV vaccines from field isolates and further enabled the genotyping of capripoxviruses isolates. Out of 61 samples tested, we identified 4 SPPV vaccines, 14 SPPV field isolates, 11 GTPVs and 32 LSDVs. The two assays were both sensitive and specific and in agreement with the se