Background and Aims: A multiplex transcription-polymerase chain reaction (m-PCR) was developed for direct detection and discrimination between Canarypox virus (CPV) and other avian poxvirus (APV).Materials and Methods: Three compatible primer sets were designed for m-PCR amplification of different loci; fpv126, fpv140, and fpv167 located at highly conserved APV genes.Results: Results showed that m-PCR products of the expected sizes were obtained for all of the primer sets when they were tested either alone or in combination with an artificial mixture of positive controls. Based on the better amplification of fpv167 than other loci, the locus primer set was used to examine tissue samples from canaries clinically diagnosed as AVP-infected.Conclusion: All Canary samples were positive for CPV by the m-PCR and virus isolation. The results of the present study indicate the m-PCR assay holds potential to be versatile, rapid, and sensitive for detection of CPV and differentiation of the virus from the other APVs.