We present the first molecular analysis of the Bufo bufo (Linnaeus, 1758) complex population in the Levant. This complex contains four species distributed through the Western Palearctic. Based on the analysis of the widely used mitochondrial marker 16S rRNA, populations from the Levant belong to B. verrucosissimus (Pallas, 1814) and have a close affiliation to populations from the Transcaucasian region rather than southern Turkey. Such identified relationships may imply past rapid colonisation from north to south and support the need for further research.