Characidium comprises several species of small freshwater fish that display conserved diploid chromosome numbers and karyotypic formulae. In this study, a comparative cytogenetic analysis using telomeric DNA probes was carried out in nine species of Characidium; a molecular phylogenetic analysis with mitochondrial DNA was also performed in order to investigate the direction of the evolutionary chromosome changes observed here. Our results showed the existence of species with several and variable interstitial telomeric sites (ITSs), with other species showing only terminal signals in their chromosomes. Molecular phylogenetic data suggested that these ITSs emerged once in the evolutionary history of Characidium and were later differentially spread in distinct species/populations of this clade. Additionally, the origin of an exclusive acrocentric pair found in C. pterostictum, C. serrano and C. timbuiense was also investigated, revealing that this pair possibly had a common origin to these species. These results evidence the occurrence of intense and continuous genomic changes among species of Characidium.