Success in smallpox eradication was enabled by the absence of non-human reservoir for smallpox virus. However, other poxviruses with a wider host spectrum can infect humans and represent a potential health threat to humans, highlighted by a progressively increasing number of infections by (re)emerging poxviruses, requiring new improved diagnostic and epidemiological tools. We describe here a real-time PCR assay targeting a highly conserved region of the poxvirus genome, thus allowing a pan-Poxvirus detection (Chordopoxvirinae and Entomopoxvirinae). This system is specific (99.8% for vertebrate samples and 99.7% for arthropods samples), sensitive (100% for vertebrate samples and 86.3% for arthropods samples) and presents low limit of detection (< 1000 DNA copies/reaction). In addition, this system could be also valuable for virus discovery and epidemiological projects.