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Western Australian sandalwood ( Santalum spicatum) is widespread throughout Western Australia across the semiarid and arid regions. The diversity and phylogeographic patterns within the chloroplast genome of S. spicatum were investigated using restriction fragment length polymorphism analysis of 23 populations. The chloroplast diversity was structured into two main clades that were geographically separated, one centred in the southern ( semiarid region) and the other in the northern ( arid) region. Fragmentation due to climatic instability was identified as the most likely influence on the differentiation of the lineages. The lineage in the arid region showed a greater level of differentiation than that in the southern region, suggesting a higher level of gene flow or a more recent range expansion of sandalwood in the southern region. The phylogeographic pattern in the chloroplast genome is congruent with that detected in the nuclear genome, which identified different genetic influences between the regions and also suggested a more recent expansion of sandalwood in the southern region.