The giant Tasmanian freshwater crayfish Astacopsis gouldi prized by fisherman, is the world's largest freshwater invertebrate. Astacopsis gouldi is known only from river drainages in northern Tasmania. A narrow distribution, pollution of habitat and over-harvesting has led to the rapid decline of populations and subsequent loss from a number of drainages. We collected mitochondrial DNA sequences to assess population structure and genetic diversity from throughout the species' distribution. We found a lineage from north-eastern Tasmania, which was genetically divergent compared with the remaining distribution in north-western Tasmania. Populations from the remaining distribution, including haplotypes found across a noted faunal barrier (Tamar River), were genetically homogeneous with <1% sequence divergence. This finding is concordant with a hypothesis of more interconnected drainages associated with lower sea levels in the past. The new cryptic lineage from north-east Tasmania requires further investigation and may be of extremely high conservation value. Conservation efforts for A. gouldi, combining habitat restoration with in situ management of wild populations and some population augmentation into once occupied rivers, will also have a positive impact for conservation of freshwater ecosystems in northern Tasmania.