Galaxias auratus and G. tanycephalus have a restricted distribution in Tasmanian highland lakes and were probably derived from a landlocked population of the diadromous G. truttaceus during the last 100,000 years. We have analysed the mitochondrial DNA and the allozyme products of 22 nuclear loci for each lake species. Four mitochondrial DNA haplotypes, defined by 40-42 six-base restriction sites, were identified amongst 26 G. auratus and three haplotypes, defined by 38-39 restriction sites, were found in six G. tanycephalus. The evolutionary genetics of reproductive isolation appears to have inflated mitochondrial but not nuclear DNA diversity between the lake species and their hypothesized progenitor, G. truttaceus. The amount of intraspecific mitochondrial and nuclear DNA diversity for G. auratus and G. tanycephalus is consistent with their having an extended and independent evolutionary history in reproductive isolation.