The fragmented distribution of Nautilus pompilius and its biology suggest there will be significant genetic divergence and spatial subdivision between east and west Australian populations. Samples were collected from the northern Great Barrier Reef, the Coral Sea, and the Scott Reef off Western Australia. Phylogenetic trees and a minimum spanning tree were developed from these data to elucidate evolutionary relationships. These data demonstrate significant evolutionary separation of each of the three populations into strongly supported discrete clades matching geographic stratification. Within each of the discrete populations, genetic variation is evident. Strong inter-population variation is evident, with discrete geographic clades being recognized for each extant group. The distinct spatial subdivision between east and west Australian populations of Nautilus are related to geographic and physical isolation over evolutionary time, and this has important fisheries management implications. The distinct geographic patterns of genetic structuring demonstrated by these data indicate the existence of discrete eastern and western Australian management units, and as such, these Nautilus populations should be managed discretely, as each has a high conservation value containing unique genetic variation.