There are two recognized poxviruses that are associated with disease in tree squirrels: squirrel fibroma virus (SQFV), Leporipoxvirus, which affects eastern grey squirrels (Sciurus carolinensis) in eastern North America, and squirrelpox virus (SQPV), a member of a newly identified poxvirus genus, which affects European red squirrels (Sciurus vulgaris) in the United Kingdom. In August 2008, a cutaneous poxvirus-associated disease was identified in a North American red squirrel (Tamiasciurus hudsonicus) from the Yukon Territory, Canada. The gross and microscopic appearance of the skin lesions was more consistent with SQPV than SQFV, and electron microscopy revealed poxvirions only within epithelial cells. Polymerase chain reaction (PCR) was used to identify poxvirus core protein encoding DNA in skin samples, and phylogenetic analysis showed that the inferred amino acid sequence was distinct from all other poxvirus species for which the core protein gene has been sequenced, including those of the genus Leporipoxvirus. Although the core protein sequence of SQPV was not available, comparison of the constructed phylogenetic tree to other published trees, based on major outer envelope proteins, revealed that the identified sequence occupies a position similar to SQPV in terms of its relationship to other poxviruses. However, PCR primers designed to amplify gene sequences encoding the SQPV major envelope protein and RNA polymerase did not amplify any sequences from infected tissues. These findings suggest that the virus present in this squirrel is a novel poxvirus of North American red squirrels. To our knowledge, this is the first case of poxvirus infection in Canadian squirrels outside of Ontario.