COI sequence variation in groundwater amphipods of Western Australia showed multiple highly divergent lineages associated with tributaries. Estimates of the timing of this diversification are consistent with the hypothesis that aridification of the region during the Tertiary forced freshwater fauna into subterranean refugia. The groundwater isopod Pygolabis provides an opportunity to compare phylogenetic signals in co-distributed taxa that might share similar histories. A 549 bp fragment of COI was sequenced for 135 individuals of Pygolabis from 12 sites. Phylogenetic analyses revealed 12 highly divergent lineages associated with tributary boundaries. Lineages were separated by 11.8–27.8% sequence divergence. This level of divergence is consistent with diversification in the Pliocene, supporting the aridification hypothesis. While lineages were associated with distinct tributaries in both amphipods and Pygolabis, the two groups showed different phylogenetic patterns, suggesting that the mode of colonisation of the groundwater differed between the amphipods and Pygolabis. ©2009 Elsevier Inc. All rights reserved.