Mitochondrial DNA (mtDNA) sequences were used to evaluate phylogeographic structure within and among populations of three endangered Hawaiian tree snail species (n = 86). The primary focus of this investigation was on setting conservation priorities for Achatinella mustelina. Limited data sets for two additional endangered Hawaiian tree snails, A. livida and A. sowerbyana, were also developed for comparative purposes. Pairwise genetic distance matrices and phylogenetic trees were generated, and an analysis of molecular variance was performed on 675-base pair cytochrome oxidase I gene sequences from multiple populations of Hawaiian tree snails. Sequence data were analysed under distance-based maximum-likelihood, and maximum-parsimony optimality criteria. Within the focal species, A. mustelina, numbers of variable and parsimony informative sites were 90 and 69, respectively. Pairwise intraspecific mtDNA sequence divergence ranged from 0 to 5.3% in A. mustelina, from 0 to 1.0% in A. livida and from 0 to 1.9% in A. sowerbyana. For A. mustelina, population genetic structure and mountain topography were strongly correlated. Maximum genetic distances were observed across deep, largely deforested valleys, and steep mountain peaks, independent of geographical distance. However, in certain areas where forest cover is presently fragmented, little mtDNA sequence divergence exists despite large geographical scales (8 km). Genetic data were used to define evolutionarily significant units for conservation purposes including decisions regarding placement of predator exclusion fences, captive propagation, re-introduction and translocation.