Infections caused byPenicilliumspecies are rare in dogs, and the prognosis in these cases is poor. An unknown species ofPenicilliumwas isolated from a bone lesion in a young dog with osteomyelitis of the right ilium. Extensive diagnostic evaluation did not reveal evidence of dissemination. Resolution of lameness and clinical stability of disease were achieved with intravenous phospholipid-complexed amphotericin B initially, followed by long-term combination therapy with terbinafine and ketoconazole. A detailed morphological and molecular characterization of the mold was undertaken. Sequence analysis of the internal transcribed spacer revealed the isolate to be closely related toPenicilliummenonorumandPenicilliumpimiteouiense. Additional sequence analysis of β-tubulin, calmodulin, minichromosome maintenance factor, DNA-dependent RNA polymerase, and pre-rRNA processing protein revealed the isolate to be a novel species; the namePenicilliumcanissp. nov. is proposed. Morphologically, smooth, ovoid conidia, a greenish gray colony color, slow growth on all media, and a failure to form ascomata distinguish this species from closely relatedPenicilliumspecies.