The dispersal of freshwater fishes in the Cape Floristic Region of South Africa has been attributed to river capture events and confluence of rivers during sea level regression. The role of low drainage divides and inter-basin water transfers have received less attention. A unique lineage of Galaxias zebratus (hereafter the Joubertina galaxias) occurs in two currently isolated river systems, the Gamtoos and Krom. The present study mapped the distribution of the Joubertina galaxias and used mitochondrial and nuclear DNA sequences to assess the processes that could have influenced its current distribution pattern. Analyses of both mitochondrial cytochrome ♭ and nuclear (S7) sequences revealed that observed genetic differentiation cannot be explained by isolation between the Gamtoos and Krom River systems. No genetic differentiation was found between the Krom River System and the Twee River (a tributary of the Gamtoos River System). Shallow genetic differentiation (0.4% for cytochrome ♭ and 0.3% for S7) was found between the Krom and the remaining populations in the Gamtoos River System. High levels of genetic structuring were observed within the Gamtoos River System with most tributaries having one or more unique alleles. Inter-basin dispersal during pluvial periods or recent human mediated translocation seems to be the most plausible explanations for presence of the Joubertina galaxias in the Krom River System. The present study also assessed the threats and habitat preferences of the Joubertina galaxias, and an evaluation of the conservation status of this lineage was done. The Joubertina galaxias is threatened mainly by alien fish invasion, habitat loss and loss of genetic diversity due to fragmentation of its populations. Due to its very restricted geographic range, small known population sizes and the intensity of threats to this lineage’s survival, this lineage has been assessed as Endangered. The lineage has a restricted Area of Occupancy (AOO) and Extent of Occurrence (EOO). The extent of occurrence has declined in all tributaries and is continuing to decline in all except two tributaries that are secure. The lineage may have had natural fragmentation among its populations, but recent threats have completely isolated the populations. The threats affect the lineage’s survival potential in the four tributaries which have small populations that are not presently viable. The densities observed for the Joubertina galaxias ranged from 0.16 - 1.3 fish /m² and the number of mature individuals for the whole population seems to be less than 2500 mature individuals. There is therefore a need for specific conservation actions to ensure the long-term survival of this unique lineage.