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Neotropical fishes exhibit remarkable karyotype diversity, whose evolution is poorly understood. Here, we studied genetic differences in 60 individuals, from 11 localities of one species, the wolf fish Hoplias malabaricus, from populations that include six different "karyomorphs". These differ in Y-X chromosome differentiation, and, in several cases, by fusions with autosomes that have resulted in multiple sex chromosomes. Other differences are also observed in diploid chromosome numbers and morphologies. In an attempt to start understanding how this diversity was generated, we analyzed within- and between-population differences in a genome-wide sequence data set. We detect clear genotype differences between karyomorphs. Even in sympatry, samples with different karyomorphs differ more in sequence than samples from allopatric populations of the same karyomorph, suggesting that they represent populations that are to some degree reproductively isolated. However, sequence divergence between populations with different karyomorphs is remarkably low, suggesting that chromosome rearrangements may have evolved during a brief evolutionary time. We suggest that the karyotypic differences probably evolved in allopatry, in small populations that would have allowed rapid fixation of rearrangements, and that they became sympatric after their differentiation. Further studies are needed to test whether the karyotype differences contribute to reproductive isolation detected between some H. malabaricus karyomorphs., (© 2024. The Author(s), under exclusive licence to The Genetics Society.)