1. Analysis of mitochondrial and nuclear DNA (microsatellites) in 379 individuals, collected from 15 localities in northern Australia and Papua New Guinea (PNG), demonstrated that wild redclaw crayfish (Cherax quadricarinatus) populations consist of two highly divergent Australian lineages and two PNG lineages.
2. The disjunction between the two Australian lineages occurs over a distance of approximately 200 km in the south-western corner of the Gulf of Carpenteria. These data conflict with an earlier study that detected no significant differentiation in 23 variable allozyme loci in redclaw sampled from northern Australia, but concur broadly with the previous recognition of two morphologically distinct species (C. quadricarinatus and C. bicarinatus) across northern Australia, and a third species in PNG (C. albertsii).
3. The inferred timing and patterns of divergence evident in the molecular data presented here closely align with a similar pattern reported in a co-distributed freshwater decapod crustacean, and broadly reflect patterns in some vertebrate taxa with similar distributions across northern Australia and PNG.
4. These congruent patterns most probably reflect periodic Plio-Pleistocene land and freshwater connections between Australia and New Guinea.