The family Gasteropelecidae is composed of three genera and eight species. This study shows that Thoracocharax cf. stellatus has 2n = 52 chromosomes for both sexes. The five males studied showed 8 metacentric, 16 submetacentric, 4 subtelocentric, and 24 acrocentric chromosomes; the seven females showed only one submetacentric chromosome, belonging to pair 11, and one extra acrocentric chromosome, smaller than all the other chromosomes, characterizing the presence of a ZZ/ZW sex chromosome system in this species. Nucleolus organizing regions (NORs) were detected on the short arms of the subtelocentric chromosome pair 13. Constitutive heterochromatin was identified at pericentromeric and terminal positions in almost all chromosomes. The W chromosome was almost entirely heterochromatic, except for a small terminal euchromatic segment. The analyses of the amount of nuclear DNA found 2.18 ± 0.09 pg of DNA per diploid nucleus, without significant differences between sexes. A discussion about the evolution of the sex chromosomes in this group is presented.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)