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Eighteen Barbus samples from the Sahara Desert territory of Morocco, Algeria and Tunisia were studied by allozyme electrophoresis. Initially, eight different taxa were identified. Five species of the genus Barbus are recognised in the Sahara Desert: B. antinorii only known from small wells in the Tunisian desert, B. biscariensis widely distributed through the Algerian desert and the Biskra Mountains, B. massaensis in west Morocco from the Massa basin to the Tamrhakht basin, B. pallaryi from the Zousfana basin and springs with thermal waters around the Figuig oasis and B. lepineyi grouping populations of the Noun, Dra and Ziz basins. Two main lineages can be differentiated: B. pallaryi, B. lepineyi and B. massaensis from the Western Sahara, and B. biscariensis from the Eastern Sahara. The phylogenetic relationships of B. antinorii are an exception to the model. This last species inhabiting the Eastern Sahara is closer phylogenetically to Western Saharan species. A tentative model of double colonisation is proposed for southern Atlas Saharan barbels as a result of these phylogenetic relationships. In the Western Sahara, two ancient hydrographic systems are postulated. The first, located in the most westerly region, is currently formed by the basins of the rivers Dra, Noun and Ziz. The second, situated in eastern Morocco and western Algeria, is currently formed by the Zousfana basin. The separation of these two hydrographic systems occurred during the Upper Pliocene (1.7 my). The time of separation between B. callensis from northern Algeria and Tunisia and B. biscariensis and B. antinorii south of these countries contradicts the classical hypothesis of a European origin for North African barbel and colonisation through Spain during the Messenian crisis and supports the hypothesis of a more ancient origin for African barbels and a southern colonisation of the Iberian Peninsula for this evolutionary branch of limnophilous barbel.