Hoplias malabaricus is one of the most widespread freshwater fish in the Neotropics; all karyotyped populations exhibit diploid numbers ranging from 2n = 39 to 2n = 42, with variations that make up to seven karyomorphs, suggesting the existence of a species complex or paraspecies. In this study, we explored the diversity of H. malabaricus of the Magdalena River Basin using karyotypic analysis. Cytogenetic analyses were carried out on 32 specimens and six sample sites from the upper, middle, and lower Magdalena River, a trans-Andean basin in Colombia, using standard and molecular techniques and fluorescence in situ hybridization (FISH). The upper and middle Magdalena samples were 2n = 42 and showed unique fluorescence patterns, whereas the lower Magdalena samples had 2n = 40, generalized fluorescence patterns, and one specimen with a mosaic karyotype and heteromorphic chromosome pair. The genetic discontinuity along the Magdalena River fits the museum hypothesis, where the 2n = 42A predates the modern configuration of the Magdalena Basin in the Late Miocene, and the 2n = 40C karyomorph represents a more derived condition. Our results underline the complex biogeography of the trans-Andean region, where the Magdalena-Cauca Basin should be considered a freshwater fauna mosaic with divergent evolutionary affinities.