Dendritic channel patterns have the potential to isolate populations within drainages, depending on the relative position within the stream hierarchy of the populations. We investigated the extent of genetic subdivision in the Australian freshwater fish Pseudomugil signifer (Kner) (Pseudomugilidae) from two drainages in northern Queensland, Australia, using allozyme techniques. The drainages were adjacent and had similar channel patterns each with two major subcatchments coalesced to an estuarine confluence. Analysis of 30 sites across the two drainages revealed that although there was significant genetic variation among sites in both drainages, this was not between the two subcatchments in either case. This result did not support predictions of the stream hierarchy model (SHM), which would predict higher levels of variation among subcatchments than within them, nor did it suggest that estuarine conditions represent a significant barrier to dispersal in this species. More variation was among sites within each subcatchment. Multidimensional scaling plots revealed that, although most sites within a drainage were similar to one another, outlier sites occurred in each drainage, so correlations between genetic distance and geographic distance were weak. We suggest that the distance between sites and the probability of connectivity between sites may better explain the observed distribution of genetic diversity.