The genetic structure of disjunct populations is determined by founding genetic properties, demographic processes, gene flow, drift and local selection. We aim to identify the genetic consequences of natural population disjunction at regional and local scales in Hakea oldfieldii using nuclear and plastid markers to investigate long-term effective population sizes and gene flow, and patterns of diversity and divergence, among populations. Regional divergence was significant as shown by a consistent pattern in principal coordinates, neighbor-joining and Bayesian analyses, but divergence at the local level was also significant with localized distribution of plastid haplotypes and populations clustering separately in Bayesian analyses. Historical, recent and first-generation gene flow was low, suggesting that recent habitat fragmentation has not reduced gene migration significantly. Genetic bottlenecks were detected in three populations. Long-term effective population size was significantly correlated with the number of alleles/locus and observed heterozygosity, but not with census population size, suggesting that the loss of diversity is associated with long-term changes rather than recent fragmentation. Inbreeding coefficients were significant in only three populations, suggesting that the loss of diversity is linked to drift and bottlenecks associated with demographic processes (local extinction by fires) rather than inbreeding. Historical disjunction as a result of specific ecological requirements, contraction of habitats following drying during the Pleistocene, low gene flow and changes in population size are likely to have been important forces driving divergence through isolation by distance and drift.