Very high rates of extinction are recorded in freshwater ecosystems, with coastally distributed species threatened by urban development, pollution and climate change. One example, the world's second smallest freshwater crayfish (genus Tenuibranchiurus), inhabits coastal swamps in central-eastern Australia. Although only one species is described (Tenuibranchiurus glypticus), it was expected that populations isolated through habitat fragmentation would be highly divergent. The aims of this study were to determine if populations of Tenuibranchiurus are genetically distinct, and if ancient divergence, as indicated in other species in the region, was evident. Tenuibranchiurus were collected at seven sites, extending the known geographical distribution ,260 km south to Wooli, New South Wales. Analysis of two mitochondrial DNA gene regions indicated two highly divergent clades, with numerous additional subclades. Both clades and subclades were strongly congruent with geographical location, and were estimated to have diverged from each other during the Miocene or Pliocene era. Little sharing of haplotypes between subpopulations was evident, indicating negligible gene flow, and genetic differentiation between subclades possibly indicates distinct species. The coastal distribution of Tenuibranchiurus, severe habitat fragmentation and clear differences between subclades suggest that they should be recognised as evolutionarily significant units, and be treated as such if conservation and management initiatives are warranted.