Diversity in Australia's freshwater fish fauna is relatively depauperate when compared to other landmasses. However the family Melanotaeniidae is one of Australia's most widespread and speciose groups of freshwater fishes. As such, they are an ideal group in which to examine freshwater phylogeography within Australia, as they offer the opportunity to compare species with different niches, but similar evolutionary history. This dissertation investigates patterns of genetic diversity in three species of Australian freshwater rainbowfishes, two co-distributed species with distinct niches in undisturbed habitat, and one species in an urban habitat, and explores the historical and contemporary processes that have influenced them. In my first chapter I used two co-distributed Melanotaenia species to test the hypothesis that a widespread habitat generalist will have lower levels of genetic diversity and population structure than a closely related habitat specialist. I used sequence from one mitochondrial gene and one nuclear gene to investigate patterns of genetic diversity in M. splendida and M. trifasciata and to determine how differences in habitat preference and historical changes in drainage boundaries have affected patterns of connectivity and isolation. M. splendida, a widespread species found in the vast majority of freshwater habitats in northern Australia, showed high levels of genetic diversity, and very little population structure across its range. Conversely, M. trifasciata, having a greatly contracted distribution to the northernmost rivers of Queensland and the Northern Territory and habitat preference for faster flowing, highly oxygenated upland streams, showed extremely high levels of population structure. While phylogeographic patterns differed, both showed a strong relationship between stream length and genetic distance. For M. trifasciata genetic distance was best explained by stream length within catchments, and an ocean distance at 100x coast length, likely reflecting infrequent dispersal between catchments at times of low sea level (r 2 = 0.82). M. splendida had a much shallower relationship with geographic distance, and genetic distance was best explained by stream length and a weaker ocean distance (10x coast length), suggesting greater rates of gene exchange. These results suggest that, although these species are co-distributed they appear to have experienced different evolutionary histories, with differences in habitat preference within waterways resulting in contrasting scales of genetic patterns. In chapter two I identified hybrid zones between co-distributed M. splendida and M. trifasciata at the periphery of M. trifasciata's distribution. I used morphological identification, mtDNA sequences and two nuclear single nucleotide polymorphism (SNP) diagnostic restriction assays to characterize incidence, levels and directionality of gene flow between these two reciprocally monophyletic taxa. Four populations were identified as having undergone extensive hybridization between M. splendida and M....