Major biogeographic barriers in eastern Australia have shaped the population structure of widely distributed and its putative subspecies.

We have investigated the impact of recognized biogeographic barriers on genetic differentiation of grey box (), a common and widespread tree species of the family Myrtaceae in eastern Australian woodlands, and its previously proposed four subspecies , , , and . A range of phylogeographic analyses were conducted to examine the population genetic differentiation and subspecies genetic structure in in relation to biogeographic barriers. Slow evolving markers uncovering long term processes (chloroplast DNA) were used to generate a haplotype network and infer phylogeographic barriers. Additionally, fast evolving, hypervariable markers (microsatellites) were used to estimate demographic processes and genetic structure among five geographic regions (29 populations) across the entire distribution of . Morphological features of seedlings, such as leaf and stem traits, were assessed to evaluate population clusters and test differentiation of the putative subspecies. Haplotype network analysis revealed twenty chloroplast haplotypes with a main haplotype in a central position shared by individuals belonging to the regions containing the four putative subspecies. Microsatellite analysis detected the genetic structure between Queensland (QLD) and New South Wales (NSW) populations, consistent with the McPherson Range barrier, an east-west spur of the Great Dividing Range. The substructure was detected within QLD and NSW in line with other barriers in eastern Australia. The morphological analyses supported differentiation between QLD and NSW populations, with no difference within QLD, yet some differentiation within NSW populations. Our molecular and morphological analyses provide evidence that several geographic barriers in eastern Australia, including the Burdekin Gap and the McPherson Range have contributed to the genetic structure of . Genetic differentiation among populations supports the recognition of some but not all the four previously proposed subspecies, with being the most differentiated.