Polyploidy, hybridization and variation in mating systems are central issues for a deeper understanding of animal evolution. The Iberian species Squalius alburnoides represents an example combining all three phenomena. Previous studies showed that S. alburnoides populations are mainly composed of triploid and diploid hybrid forms (mainly females), and that the tetraploid forms are rare or absent. Both populations from the Douro drainage reveal a distinct scenario: tetraploid individuals represent 85.6-97.5% of the population, with no sex ratio bias observed. Based on the flow cytometry measurements of blood and spermatozoa cells, microsatellite loci and experimental crosses, we describe here, for the first time, two symmetric allotetraploid populations (CCAA) that resumed normal meiosis after undergoing intermediate processes of non-sexual reproduction to give rise to a new sexually reproducing polyploid species. Prezygotic (habitat selection and assortative mating) and postzygotic mechanisms (nonviable embryos) are responsible for the reproductive isolation from other forms of the S. alburnoides complex (e.g. CA, CAA). This example illustrates how hybrid polyploid complexes may lead to speciation.