The Kimberley region of Western Australia possesses a poorly studied freshwater fish fauna with high endemism in an aquatic landscape subject to monsoonal floods and dry season isolation. In the first population genetic study of freshwater fish in this region, the authors tested the effects of geographic barriers on genetic structure at multiple spatial scales in east Kimberley populations of the western rainbowfish, Melanotaenia australis, the most widespread and abundant species in the region. Based on allozyme comparisons, hierarchical analysis of F(ST) revealed increasing genetic subdivision with spatial scale. Minimal genetic structure within creeklines demonstrated that wet season dispersal, rather than dry season isolation, determines genetic structure at small scales. At the scale of sub-catchments, a pattern of isolation by distance along creeklines was evident. Genetic subdivision between adjacent river systems was greater between rivers separated by a plateau than by lowlands. This implies greater connectivity of populations in lowland areas and may explain the greater similarity of the east Kimberly freshwater fish fauna with lowlands to the east than with the more rugged regions to the west. Similarly, greater connectivity between lowland populations may account for the on-average larger distribution of lowland Melanotaeniids.