Genetic diversity of Chara populations found in Israeli Negev Desert streams was analyzed using amplified fragment length polymorphism (AFLP) in 31 individuals. The adjacent streams Avdat and Aqev were used as study sites in the Ein Avdat National Park. A Jaccard phylogenetic tree, based on 468 loci, showed clear separation between four Chara species: C. vulgaris, C. contraria, C. gymnophylla and Chara sp. The last two species served as out groups from the Northern Israel. In both C. vulgaris and C. contraria, genetic differences were found between the populations originating in the two streams: higher private loci number (80 vs. 24 in C. vulgaris and 58 vs. 25 in C. contraria) and polymorphic loci level (45% vs. 23% in C. vulgaris and 39% vs. 21% in C. contraria) were found in Avdat compared to Aqev. The genetic divergence revealed within and between the two streams is presumably adaptive and determined by natural selection associated with ecological stress. Sunlight intensity, water level and pH were found to be the main ecological variables associated with species clustering, through selection, in the varying ecology of the sampling stations along each of the streams. Evidence for gene flow between and within the streams was found using the structure analysis, suggesting that sampling sites condition is the regulating factor for oospores establishing, and hence, gene flow occurs more often between sampling sites with similar ecological conditions.