The karyotypic and chromosomal characteristics of the hatchetfish Thoracocharax stellatus from the Araguaia River, Brazil (Araguaia-Tocantins basin) were analyzed using Giemsa, AgNO3, and CMA3 fluorescent staining, and C-banding. The diploid chromosome number was 54 and the karyotypes of females and males were composed of six metacentrics, six submetacentrics, six subtelocentrics and 36 acrocentrics. Two unpaired acrocentric chromosomes were detected in the female karyotype. C-banding showed heterochromatic blocks at several chromosomes and an entirely heterochromatic acrocentric chromosome in females that was lacking in the male karyotype. This discovery indicated a heteromorphic sex chromosome system of the ZZ/ZW type. Ag-staining and CMA3 fluorescence revealed one major chromosome pair bearing the NORs with the presence of additional signals in some metaphases. Both heterochromatic segments associated with Ag-NORs and the W chromosome were positively stained by CMA3. Considering the present data and previous findings it is hypothesized that the occurrence of ZW sex chromosome system is widespread in the genus Thoracocharax.