Erythrinus erythrinus, a Neotropical fish species of the Erythrinidae family, has a wide distribution in South America. Previous cytogenetic analysis showed that this species presents extensive karyotype diversity, with 4 karyomorphs (A-D) described herein. This study investigated the karyotypic structure of 2 new populations of E. erythrinus from the Brazilian Pantanal region, in order to improve the knowledge of the chromosomal diversity in this species. Both populations showed typical characteristics of karyomorph A, with 2n=54 chromosomes (6m+2st+46a), without differentiation between males and females. In addition, identical supernumerary B chromosomes, appearing as double-minute chromosomes, were also found in both populations. These findings suggest the presence of mitotic instability in view of their high intra- and inter-individual numerical variation. The presence of these chromosomes is likely a basal characteristic for this group, since the same kind of Bs also occurs in some other populations and karyomorphs of E. erythrinus. As such, they are important markers of biodiversity found in this nominal species, which probably corresponds to a species complex.