These species are living in 4 basin and are distinctive morphologically and we certain that they are 5 different species.
I would start with a mitochondrial gene, probably either COI or Cytb, depending what data is available in GenBank and/or BOLD. I would use the sequences to estimate a gene-tree to get a first insight in the relationships between the fishes. But all of this is dependent on your specific question.
At least one additional nuclear gene as Histone 3 or 28s would be desirable to check for recent (or ancient) gene flow.
Assuming the five species are known, they are likely in the GenBank data base, which will suggest which genes (likely mtDNA) to sequence to assign them to known taxa. If you don't know which species are present, again using the most extensively used gene segment will identify them by comparison with known species. If you find a new species, congratulations!
I agree with what has been previously stated about one or two mtDNA genes being a good first assessment. My colleagues and I have found cyt b and ND2 to be informative for divergences in the Pliocene and Pleistocene, and the first nuclear intron of S7 was informative for divergences in the Miocene and Pliocene. I know this would be dependent on funding and expertise of lab personnel, but at this point you'd likely be better off using sequence capture and Illumina sequencing to address your questions.
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