Spatial genetic variation within species is influenced by both contemporary and historical factors. We attempted to assess the impact of increased aridity and lower temperatures associated with the last glacial maximum on possible refugia and the structuring of genetic variation in Banksia hookeriana, a shrub species restricted to deep sands on the Eneabba sandplain and adjacent Gingin Scarp/Dandaragan Plateau, centred 300 km north of Perth, Western Australia. We used optically stimulated luminescence (OSL) dating to estimate the last time these sands were mobile, as well as amplified fragment length polymorphisms (AFLP) to infer spatial patterns and the phylogeographical history of genetic variation among 15 populations of B. hookeriana. While genetic variation at the species level was high, with 96.6% of 238 AFLP markers polymorphic, average within population gene diversity was low (Hpop = 0.16). Of the total genetic variation, an analysis of molecular variance (amova) partitioned 70% within populations, 24% among populations within substrate and 6% between substrates. There was an isolation-by-distance effect among populations within the same substrate, but not across substrates, and ordination highlighted genetic differentiation between the sandplain and scarp/plateau populations. A neighbour-joining tree identified the sandplain populations as a distinct clade, with the exception of the most northern sandplain population, which clustered with two northern and eastern plateau populations. The most southern plateau populations formed a clade sister to the sandplain clade. OSL dating of sand at six extant populations suggested that dunes were last mobile 15 000–35 000 years ago, with no clear difference in the ages of sandplain and plateau dunes. These data are consistent with a historical scenario of (re)colonization from isolated refugia of smaller populations either within the patchily vegetated sandplain and/or refugia at the northern, eastern and southern sandplain/scarp margins following postglacial climate amelioration and dune stabilization. Historic interpretations were confounded by the possible effects of long-distance dispersal, natural selection by substrate, and weak and/or ancient introgression with the sister species, Banksia prionotes.