Invasive and native populations of the Amazonian fishes 'peacock bass' Cichla monoculus and of a not yet described species 'blue tucunare' here referred as Cichla sp. 'Azul' were analysed for genetic diversity using the hypervariable domain of the mitochondrial DNA (mtDNA) control region plus steady diagnostic random amplified polymorphic DNA loci. There is no detailed historical record of the introduction of Cichla species into the Upper Parana River basin, where they became invasive and a potential threat to local ichthyofauna. Genetic diversity among invasive populations confirmed the hypothesis of multiple introductions in this hydrographic basin. Moreover, a large and previously unknown population of natural fertile hybrids between C. cf. monoculus and Cichla sp. 'Azul' was identified in the Itaipu hydroelectric reservoir and in the floodplain of the Upper Parana River. Crossbred morphotypes were similar to C. cf. monoculus, but their morphological identification was not unequivocal. This hybrid population was characterized by high genetic diversity and it was composed of hybrids possessing concurrently nuclear DNA fragments specific for C. cf. monoculus as well as fragments specific for Cichla sp. 'Azul'. The nuclear DNA markers indicated that reproductive isolation between C. cf. monoculus and Cichla sp. 'Azul' has broken down in the new environment, and mtDNA sequences revealed that both species can be the female donor in the interspecific crosses. The data presented herein are potentially useful for future taxonomic, genetic and evolutionary studies in the complex Cichla group, for monitoring of invasive populations, and for further development of ecological guidelines.