Abstract Background Among Neotropical fishes, the family Characidae is highly diverse and speciose and its taxonomy is not completely resolved. In Chile, the family is represented by five species, all within the genus Cheirodon , of which C. pisciculus, C. galusdae, C. kiliani , and C. australe are endemic , while C. interruptus is introduced. We compared chromosomal markers in order to appreciate the taxonomy and evolution of these trans-Andean fishes. Results The specimens were photographed in stereomicroscope to observe the ventral protrusive teeth and cusps for morphological analysis and species identification. All of the species analysed had equally diploid chromosome number 2n = 50, with karyotypes dominated by high number of acrocentric chromosomes as compared to those of other members of Cheirodontinae. The distribution of heterochromatin was inconspicuous and was similar in all the species. The number of active NORs (nucleolus organizer regions) was polymorphic, with the greater number of them in C. kiliani and C. galusdae . The location of 5S and 18S rDNA ranged in number and position, showing two sites in different chromosomes. The fluorescent in situ hybridization with telomeric probe did not reveal interstitial sites in all analysed species. Conclusions The comparative analysis of karyotypes and morphological markers revealed a biogeographic pattern of distribution, with the species that occur in the southern region forming one group and those in central and northern Chile forming another.