The region around the Strait of Gibraltar is considered to be one of the most relevant ‘hot spots’ of biodiversity in the Mediterranean Basin due to its historical, biogeographical, and ecological features. Prominent among these is its role as a land bridge for the migration and differentiation of species during the Pleistocene, as a consequence of the lowering of sea level and climate changes associated with the Ice Ages. In the present study, we report a multilevel hierarchical investigation of the genetic diversity of Calicotome villosa, a common pioneer legume shrub, at the regional scale. The results of genetic analysis of progeny arrays are consistent with a predominantly outcrossing mating system in all the populations analysed. Geographically, a pattern of population isolation by distance was found, but the Strait accounted for only approximately 2% of the among-population genetic differentiation. Consequently, extensive historical gene flow appears to be the rule for this species in this area. According to the natural history traits of C. villosa (pollination, dispersal, and colonization ability), we hypothesize that gene flow must be strongly influenced by seed dispersal because pollen flow is very limited. Based on the history of trade and land use, cattle and human movements across the Strait must have strongly favoured seed dispersal. We review and discuss these results and compare them with those of other reported studies of genetic and phylogenetic differentiation across the Strait of Gibraltar. It is stressed that colonization ability, which depends upon seed dispersal and life form, can be a more critical factor in gene flow than pollination. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 93, 39–51.