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Israel marks a crossroads between three continents encompassing several phytogeographical and zoogeographical zones. In this complex area, the flow of species from different biogeographical regions creates opportunities to study how geographical division and colonization routes affect current distribution and structure of resident populations of organisms associated with desert and and environments, habitats that may have persisted throughout Pleistocene glacial periods. The present paper analyses the population history of the spider Stegodyphus lineatus in the contiguous Negev and Judean deserts in Israel using allozyme and mtDNA variation. The distinct patterns of variation indicate that Judean and Negev populations are vicariant lineages. The residence time was longer in Judea, where populations were more polymorphic for mtDNA, showed isolation by distance and were less structured than in the Negev. The Negev population, possibly linked to other Mediterranean populations of S. lineatus, consisted of two subdivisions derived from a recent eastward expansion across the central Negev watershed. Despite differences in age and level of structure, all lineages show similar dispersal processes dominated by restricted gene flow. The distribution patterns of allozyme and mtDNA markers are unrelated to geographical patterns of precipitation and vegetation. Rather, they follow large-scale topographic features, namely the water divide between Mediterranean and Afro-Syrian rift drainages and between eastern and western Negev drainages. (c) 2005 The Linnean Society of London.