Fishes of the genus Carassius are useful experimental vertebrate models for study of evolutionary biology and cytogenetics. Carassius demonstrates diverse biological characteristics, such as variation in ploidy levels and chromosome numbers, and presence of microchromosomes. Carassius polyploids with ≥ 150 chromosomes have microchromosomes, but their origin, especially in European populations, is unknown. We used cytogenetics to study evolution of tandem repeats (U1 and U2 small nuclear DNAs and H3 histone) and microchromosomes in Carassius from the Czech Republic. We tested the hypotheses whether the number of tandem repeats was affected by polyploidization or divergence between species and what mechanism drives evolution of microchromosomes. Tandem repeats were found in tetraploid and hexaploid C. gibelio, tetraploid C. auratus and C. carassius in conserved numbers, with the exception of U1 snDNA in C. auratus. This conservation indicates reduction and/or loss in the number of copies per locus that may have occurred by divergence rather than polyploidization. To study evolution of microchromosomes, we used the whole microchromosome painting probe from hexaploid C. gibelio and hybridized it to tetraploid and hexaploid C. gibelio, tetraploid C. auratus and C. carassius. Our results revealed variation in the number of microchromosomes in hexaploids, indicating that the evolution of Carassius karyotype is governed by macrochromosome fissions followed by segmental duplication in pericentromeric areas. This is a potential mechanism responsible for the presence of microchromosomes in Carassius hexaploids. Differential efficacy of one or both of these mechanisms in different tetraploids could ensure variability in chromosome number in polyploids in general.