Avian poxvirus (genus Avipoxvirus, family Poxviridae) is an enveloped double-stranded DNA virus that may be transmitted to birds by arthropod vectors or mucosal membrane contact with infectious particles. We characterized the infection in Anna's Hummingbird (Calypte anna; n = 5 birds, n = 9 lesions) by conducting diagnostic tests on skin lesions that were visually similar to avian poxvirus lesions in other bird species. Skin lesions were single or multiple, dry and firm, pink to yellow, with scabs on the surface, and located at the base of the bill, wings, or legs. Microscopically, the lesions were characterized by epidermal hyperplasia and necrosis with ballooning degeneration, and intracytoplasmic inclusions (Bollinger bodies) in keratinocytes. The 4b core gene sequence of avian poxvirus was detected by PCR in samples prepared from lesions. Nucleotide sequences were 75–94% similar to the sequences of other published avian poxvirus sequences. Phylogenetic analyses showed that the Anna's Hummingbird poxvirus sequence was distinguished as a unique subclade showing similarities with sequences isolated from Ostrich (Struthio camelus), Wild Turkey (Meleagris gallopavo), falcons (Falco spp.), Black-browed Albatross (Diomedea melanophris), Mourning Dove (Zenaida macroura) and White-tailed Eagle (Haliaeetus albicilla). To our knowledge this is the first published report of definitive laboratory diagnosis of avian poxvirus in a hummingbird. Our results advance the science of disease ecology in hummingbirds, providing management information for banders, wildlife rehabilitators, and avian biologists.