Trioncinia retroflexa is an endangered endemic plant species found in the Bluegrass grasslands of central Queensland Australia. The recent degradation and fragmentation of this ecosystem is commonly assumed to have driven this species to become rare and persist only in isolated populations. Despite a lack of empirical evidence supporting this assumption (and equivocal anecdotal evidence), conservation and restoration plans for this species are generally based on it. Using microsatellites, the genetic diversity, structure and differentiation of all known populations of these species were examined to determine if there is evidence for recent isolation of remaining populations. Populations had high genetic differentiation, with little heterozygosity, minimal gene flow and few migrants, reflecting long-term population isolation well beyond the scope of modern fragmentation. High genetic differentiation also suggests that all known populations of this species maintain a similar proportion of the species’ total genetic diversity, despite varying extensively in the number of individuals they support. As such, the loss of any population may drastically reduce the species’ adaptive potential in the future. In the context of long-term population isolation, seed and germination traits related to species dispersal and establishment were examined among these long isolated populations. Though some significant differences in traits among populations were found, there was not strong evidence of trait divergence. Given the findings of this study, this species has likely persisted in isolated populations for at least the last 158 years (time since identification) and may have been rare prior to this time. A large restoration experiment completed in association with this project failed to establish a new population over a two-year period. The difficulty of establishing new populations of T. retroflexa highlights the lack of information we have about this species’ environmental requirements and the importance of maintaining the remaining natural populations of this species.