Banksia hookeriana (Proteaceae) is an endemic, fire-killed, shrub of south-western (SW) Australian Mediterranean-type shrublands restricted to acid sandplains and dunes over a geographic range of only 80 × 30 km. Amplified fragment length polymorphism (AFLP) markers were used to characterize genetic variation within and among six populations encompassing the species extant geographic range. Populations from the north, east, and south of the species range were located on deep sands over laterite on the Gingin Scarp (90-180 m a.s.l.), while three populations were on lowland dunes of the Eneabba Plain (50-80 m a.s.l.). The geomorphic history of the acid sands upon which the species occurs is poorly understood. Regardless of their origins, at least some of these sands may have been mobile during the arid phase of the last glacial-interglacial cycle. A high level of genetic variation was detected, with 94.4% of 231 AFLP markers polymorphic. Significant genetic differentiation among most populations suggested an overall low level of gene flow and the accumulation of genetic differences within relatively genetically isolated populations. A phylogeographic analysis produced a clear dichotomy between upland and lowland populations. Preliminary interpretation suggests that populations on the Eneabba Plain may have expanded there from refugia on the Gingin Scarp fǒllowing postglacial climate amelioration and dune stabilization. However, the tree is unrooted and differences between upland and plains populations may also reflect different selection pressures reflecting habitat variation.