Comparative cytogenetic studies carried out in two populations of Characidium cf. gomesi from Botucatu region, SP, Brazil, showed a similar karyotypic structure in a diploid number of 50 chromosomes, 32 metacentric and 18 submetacentric chromosomes for males and 31 metacentric and 19 submetacentric chromosomes for females as well as a ZZ–ZW sex chromosome system. Differences between both populations, however, were found in relation to the occurrence of B chromosomes and the distribution of 18S and 5S ribosomal DNA (rDNA) sites. Characidium cf. gomesi from the Alambari Stream, a component of the Tietê River basin, revealed 18S rDNA on Z and W chromosomes, while this gene was located on autosomes in the sample from the Paranapanema River basin. The 5S rDNA sites were observed in a single chromosomal pair (number 25) in the populations from Paranapanema and in two pairs in the specimens from Tietê (numbers 20 and 25). Besides that, in the sample from Paranapanema, both inter and intra-individual variations were found due to the occurrence of up to four heterochromatic supernumerary chromosomes in the cells. The life mode of this fish, restricted to headwaters and subjected to frequent breakdown into sub-populations, may have contributed to the fixation of such chromosomal differences. The karyotypic similarities found in the analysed populations, however, suggest that all are descended from the same ancestor group whereas their differences indicate that they are already existing in reproductively isolated populations.