Dispersal can play an important role in the genetic structuring of natural populations. In this regard, freshwater fishes often exhibit extensive population genetic subdivision and are ideal subjects for investigating current and historical patterns of connection and dissociation between drainages. We set out to generate a comprehensive molecular phylogeny for a widespread freshwater fish from eastern Australia, the Pacific blue-eye Pseudomugil signifer. Although movement via flood events may be important in the southern end of the species’ range, genetic structuring revealed the importance of historical drainage connections and dissociations in mediating or disrupting dispersal. A dominant feature of our phylogeny is a split between northern and southern populations, which appears to be congruent with a biogeographical barrier recently implicated as important for the connectivity of freshwater organisms in eastern Australia. The extent of the split also has taxonomic implications consistent with suggestions that the Pacific blue-eye may represent more than a single species.