A cytogenetic study of Stephanolepis setifer by conventional Giemsa stain and localization of Nucleolus Organizer Regions (NORs) by silver staining is reported. Females possess a diploid chromosome number of 2n=34, all of which are acrocentric, Males exhibit a karyotype with 2n=33 chromosomes, consisting of 32 acrocentric chromosomes including two unpaired acrocentric chromosomes, and one medium-size metacentric chromosome. The results suggest the presence of a multiple sex chromosome system of X1X1X2X2/X1X2Y type. The X1 and X2 chromosome sizes suggest that the metacentric chromosome is derived from Robertsonian fusion between the original Y chromosome and an autosome which probably corresponded to one element of the pair 16 and another of the pair 17, resulting in a neo-Y sex chromosome. Silver staining of metaphase spreads revealed a single pair of Ag-NORs interstitially located on the chromosome pair numbered 7. The comparison of our results with previous reports in Tetraodontiformes species suggest that its evolutionary divergence has been accompanied by macrostructural karyotype changes as far as microstructural alteration in NORs cluster.