Cytogenetic studies were performed in two syntopic species of Characidium, C. lauroi and Characidium sp. cf. C. alipioi, from Ribeirão Grande, Paraíba do Sul river basin. Both species have diploid number 2n=50 chromosomes, but differ in chromosome shape, C-banding pattern and location of nucleolar organizing regions. In Characidium sp. cf. C. alipioi a new type of ZW sex chromosome system composed of equal sized metacentric chromosomes is reported for the first time in the genus Characidium. Species of Characidium with a sex chromosome system form a monophyletic group. Variations in this system are interpreted as resulting from geographic isolation among allopatric species.