Annual crop fields are short-lived and disturbed environments. Therefore, sustainable populations of natural enemies in these fields must be maintained by repeated colonization each season from habitats outside the crop fields. In desert agroecosystems, unmanaged habitats differ greatly in abiotic and biotic conditions from croplands, creating potentially significant barriers to movement of predators. We asked here: to what extent do predators use non-crop habitats as refuges or breeding sites in the desert agroecosystem of the northern Negev, Israel? We investigated the use of natural desert habitat, planted trees (Eucalyptus), and a summer crop (sunflowers) by winter-wheat inhabiting spiders. We collected spiders using pitfall traps and a suction device from wheat fields and adjacent to non-wheat habitats during the wheat season and between seasons. We found that two crop specialist species, Trichoncoides piscator (Simon, 1884) (Linyphiidae) and Thanatus vulgaris Simon, 1870 (Philodromidae), switched to an alternative crop during the inter-wheat season. Habitat generalist species, such as Nomisia sp. (Gnaphosidae), Enoplognatha spp. (Theridiidae) and Alioranus pastoralis (O. Pickard-Cambridge, 1872) (Linyphiidae) used alternative non-crop habitats as refuges and breeding sites to differing degrees in both seasons. While all habitat generalist species used the desert habitat, none used planted trees exclusively as an alternative habitat. We conclude that crop-inhabiting, desert species may be unable to colonize the wheat fields if nearby desert habitat is supplanted by other crops or by tree plantations.