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The Neratva river basin is known for the high diversity of endemic freshwater fishes. A single species of spined loach, Cobitis narentana, was recorded from this area. Data about its distribution are very scarce.Until now C. narentana had been recorded only from the main flow of the Neretva River in its middle and lower parts, including the delta and in the Matica River near Vrgorac.In July 2004, the inflows of the River Neretva and surrounding karstic fields were sampled, and new data on spined loaches in the area gathered. Spined loaches were also found in the Bregava and Trebišnjica River, Matica River in Imotsko polje, Lištica River in the Mostarsko blato and in Hutovo blato wetland. However, they were not found in karstic fields situated more westerly-Livanjsko, Duvanjsko, Kupreško and Glamočko polje. Surprisingly, study of mtDNA variability revealed that loaches from the Neretva basin do not form a monophyletic lineage, but two distinct evolutionary units within the Adriatic spine loach lineage.The indicates that the species diversity of spined loaches in the Neretva basin is underestimated and a detailed taxonomic study is needed to resolve this issue.