The phylogenetic relationships of the genus Squalius are believed to be well established based on the mitochondrial cytochrome b gene. Here, we inferred the phylogenetic relationships of all species inhabiting most of the western Iberia river systems using a nuclear multi-locus approach and different species tree methods: concatenation and coalescent-based methods (BEST and minimize-deep-coalescence). The dataset comprised sequences of seven coding and three non-coding regions belonging to seven nuclear genes, which were chosen to cover multiple biological functions: amh, bmp4, ef1a, egr2, irbp, rh and rpl8. We provide evidence for a conflicting topology between the nuDNA species tree and the widely reported mtDNA gene tree. S. pyrenaicus is rendered paraphyletic in all nuDNA species trees, with populations of the Tagus/Colares clustering with S. carolitertii, while populations from the Guadiana, Sado and Almargem form a separate clade. Although a larger sampling size encompassing the full spectrum of Squalius populations in western Iberia is still needed to fully elucidate the phylogeography and species delimitation of this genus, our results suggest that the two S. pyrenaicus clades may represent different species.