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The genus Astyanax is one of the most numerous of the family Characidae, comprising a large number of similar-shaped species, but displaying innumerable karyotypic variations in its chromosome number and/or structure. The literature describes A. fasciatus populations with diploid chromosome numbers varying from 2n = 45 to 2n = 48. In this study, A. fasciatus specimens captured in the Araguari River (Alto Paraná basin) were cytogenetically characterized, revealing a diploid chromosome number of 2n = 46. The nucleolar organizing regions (NORs), detected with silver nitrate staining, showed a multiple system with two pairs of marked chromosomes. These findings are congruent with those of other studies involving populations of the same species.