Four novel phylogenetic clades of canine rabies virus (RABV) variants have been identified in the Middle East and North Africa. The three novel Middle Eastern clades comprise RABV isolates from the borders between Israel and neighbouring countries. The North African clade (Africa 4) comprises four RABV isolates from Egypt and one from Israel. We characterized various RABV lineages antigenically by using a panel of monoclonal antibodies to the nucleoprotein (N) and phylogenetically by analysis of entire N gene sequences. The estimated mean substitution rate for the N gene alignment (2.7x10(-4) substitutions per site per year) is comparable with previous estimates for RABV. The application of a molecular clock indicates the emergence of current canine RABV diversity to have occurred at about the same time (approx. 1870) in the Middle East and Europe, following divergence from established lineages in Africa and Asia.