The genus Psalidodon Eigenmann, 1911 (Characiformes Characidae) is a fish group with great diversity, expressed at the chromosomal, genetic and taxonomic level. The genus is markedby events of allopatric and vicariant evolution, by the formation of complexes of species andby wide geographical distribution. Both in these fish and other organisms, the association ofstudies with molecular markers and geometric morphometric techniques are useful in delimitingsignificantly evolutionary units (ESU). In this work, we performed maximum likelihood esti-mates (MLE) from mitochondrial Cyt b gene sequences and canonical variables (CVA) from13 landmarks in eight populations of P. aff. paranae Eigenmann, 1914. The analysis of Max-imum likelihood resulted in the structuring of populations in two different clades, one of whichwas composed only of individuals from a small population inhabiting a stream with approxi-mately two km of length, demonstrating their clear distinction from the other populations. Theanalysis of canonical variation demonstrated the complete structuring of this population, andthe position of each clade in the morpho-space was congruent with the topography observedin the MLE. Based on the results found, the existence of a new endemic species of the genusPsalidodon is evident.