In the past decades, sporadic cases of ocular Onchocerca infection have been reported in canids in US and Europe. The present study was undertaken to provide a detailed description of the morphologic characteristics of adults and microfilariae and to characterize the 5S ribosomal rRNA gene (5S rDNA) spacer sequences of Onchocerca lupi causing canine onchocercosis. The morphology of O. lupi is unique within the genus, and morphology based cluster analysis indicates that O. lupi is not closely related to the members of domestic cattle or horse clades occurring in North America and Europe. Similarly, the signature of the 5S rDNA spacer sequences of O. lupi does not resemble any other Onchocerca 5S rDNA spacer sequences including those of the members of domestic cattle or horse clades. The adult and microfilarial morphology and sequence signature supports the biological arguments that a distinct species, O. lupi and not O. lienalis, is responsible for canine ocular onchocercosis.