The contemporary and historical colonization capacity of an Australian freshwater fish, north-west glassfish Ambassis sp., was tested using mtDNA sequence data and six newly developed microsatellite loci in an endoreic basin in central Australia. Overall, Ambassis sp. exhibited weak genetic structure within catchments, suggesting some capacity to recolonize extirpated waterholes after disturbance. Genetic structure revealed that the historical pattern of connectivity among catchments in the Lake Eyre Basin was dramatically different from other species studied in this region. Two highly divergent clades were detected in separate catchments in the basin. mtDNA from individuals sampled in catchments north of the Lake Eyre Basin suggest that Ambassis sp. has colonized on two separate occasions from catchments in northern Australia, subsequently generating two highly divergent lineages.