Genetic divergence and speciation mechanisms of the Amazonian Engystomops frog have been inferred mainly from mtDNA sequences, microsatellite and male advertisement call. Although many aspects of this divergence remain unclear, cytogenetic analyses of Amazonian Engystomops populations are described and are compared to the relationships inferred from mitochondrial and nuclear DNA sequence data. High cytogenetic variation distinguished karyotypic patterns among the populations, even between populations which had no prezygotic isolation previously inferred from mating call analysis. Interestingly, the Puyo and Acre populations, which are in different clades, showed heteromorphic sex chromosomes (XX/XY), not identified in the other Ecuatorian populations analyzed. The analysis of a specimen collected in a site near Yasuní (Ecuador) which was cytogenetically related to the specimens from Puyo (Ecuador), was also phylogenetically closely related these specimens. In the rhodopsin nucleotide sequences, six polymorphic sites were identified and various specimens were heterozygous for them. All the data presented herein, in conjunction with those previously reported, corroborate the hypothesis that Engystomops petersi is a complex of distinct species. It also indicates that karyotypic evolution patterns may have played a substantial role in the Engystomops speciation and the occurrence of sporadic mating events between divergent evolutive lineages is discussed.