Diapoma pampeana was recently described to occur in the upper Negro basin in Uruguay and Brazil. An isolated population tentatively identified as D. pampeana from the Pando stream, a perturbed coastal drainage in Uruguay, is studied and compared under the light of morphological and molecular data to test if there is evidence to consider it as a separate species. New geographical records for the species are presented and included in the comparisons. The specimens analyzed were pooled into four groups: Pando, Santa Lucía, Middle Negro and Upper Negro. We analyzed 32 morphological characters using statistical procedures and recovered a COI-based phylogeny of different populations of D. pampeana to test if they may represent different species. Size-corrected PCA revealed that the Pando and Upper Negro groups are greatly diverging in both morphometric and meristic data along PC1 (mainly by the snout to dorsal-fin origin, dorsal to adipose-fin origins, number of longitudinal scales and predorsal scales). This deviating pattern was also obtained in a cluster analysis. The Santa Lucía and Middle Negro groups were found to be intermediate morphotypes. In contrast, molecular analyses revealed that the Pando and Upper Negro specimens resemble genetically and, thus, are placed together in the Neighbor-joining and Bayesian topologies, as part of a monophyletic Diapoma. We proposed that the Pando population, despite its deviating morphology observed, can be classified as D. pampeana. Therefore, this population constitutes a remarkable example of an isolated population that is morphologically divergent but genetically similar to the geographically most distant conspecific population.