Subspecific taxa of species complexes can display cryptic morphological variation, and individuals and populations can often be difficult to identify with certainty. However, accurate population identification is required for comprehensive conservation and breeding strategies and for studies of invasiveness and gene flow. Using five informative microsatellite markers and a Bayesian statistical approach, we developed an efficient polymerase chain reaction-based diagnostic tool for the rapid identification of individuals and populations of the 'Acacia saligna' species complex of Western Australia. We genotyped 189 individuals from 14 reference populations previously characterised based on morphology and used these data to investigate population structure in the species complex. High total genetic diversity (Ht=0.729) and high population differentiation (θ=0.355) indicated strong intra-specific structuring. With the provision of prior population information, the reference data set was optimally resolved into four clusters, each corresponding to one of the four main proposed subspecies, with very high membership values (Q>97%). The reference data set was then used to assign individuals and test populations to one of the four subspecies. Assignment was unequivocal for all test individuals from two populations of subsp. 'lindleyi' and for all but one individual of subsp. 'stolonifera'. Individuals from populations of subsp. 'saligna' and subsp. 'pruinescens' showed a degree of genetic affinity for the two subspecies in their assignments, although the majority of individuals were correctly assigned to subspecies. The diagnostic tool will assist in characterising populations of 'A. saligna', especially naturalised and invasive populations of unknown origin.