Antigenic characterisation of over 350 chiropteran rabies viruses of the Americas, especially from species reported rabid in Canada, distinguished 13 viral types. In close accord with this classification, nucleotide sequencing of representative isolates, at both the N and G loci, identified four principal phylogenetic groups (I-IV), sub-groups of which circulated in particular bat species. Amongst the North American bat viruses, there was a notable division between group I specimens associated with colonial, non-migratory bats (Myotis sp. and Eptesicus fuscus) and those of group II harbored by solitary, migratory species (Lasiurus sp. and Lasionycteris noctivagans). Certain species of Myotis were clearly identified as rabies reservoirs, an observation often obscured previously by their frequent infection by viral variants of other chiroptera. An additional group (III) apparently circulates in E. fuscus, whilst viruses harbored by both insectivorous and haematophagus bats of Latin America clustered to a separate clade (group IV). Comparison of the predicted N and G proteins of these viruses with those of strains of terrestrial mammals indicated a similarity in structural organisation regardless of host species lifestyle. Finally, these sequences permitted examination of the evolutionary relationship of American bat rabies viruses within the Lyssavirus genus.