Phylogenetic analysis of representatives from 18 wild populations of the giant freshwater prawn, Macrobrachium rosenbergii, utilising a fragment of the 16S rRNA mitochondrial gene, identified two major reciprocally monophyletic clades either side of a well-known biogeographic barrier, Huxley's line. The level of divergence between the two clades (maximum 6.2%) far exceeds divergence levels within either clade (maximum 0.9%), and does not concord with geographical distance among sites. 'Eastern' and 'western' M. rosenbergii clades have probably been separated since Miocene times. Within-clade diversity appears to have been shaped by dispersal events influenced by eustatic change.