Several Australian arid zone Acacia species are under threat because of decades of fruiting and recruitment failure that may reflect the loss of genetic diversity within small and isolated populations. We developed primers for eight microsatellite loci for Acacia carneorum and Acacia loderi. We detected high levels of clonality in each of two stands of A. carneorum (1 and 2 genets). In contrast, one stand of A. loderi was wholly clonal (1 genet), while in a second there were 30 unique genotypes. These loci allow assessment of the genetic diversity and connectedness of populations, the relative contribution of asexual reproduction to genotypic diversity and population structure, and use of paternity analysis to identify sires of seed within populations known to have set seed in past decades. This type of information may provide a basis for a recovery plan based on ‘genetic rescue’.