Iochrominae is a largely Andean clade known for its remarkable diversity of floral forms and colors. Although knowledge of chromosomal changes can provide insights into the processes underlying speciation, such data in Iochrominae are scant. We performed cytogenetic analyses to characterize chromosome number and morphology, CMA/DAPI heterochromatic bands, and distribution of rDNA sites in Iochrominae. Ancestral karyotypes were reconstructed on a newly-estimated molecular phylogeny in order to test congruence between karyotype evolution and clade differentiation. We found that, compared with its closest relatives, Iochrominae comprises species with highly symmetrical karyotypes, with no changes in base chromosome number. The common ancestor of Iochrominae was inferred to be a diploid with 2n = 24, with a karyotype with 0-2 submetacentric chromosomes and the rest metacentric, an arm ratio ca. 1.30, one locus of 45S or NORs, and one locus of 5S. Using phylogenetic comparative methods, we estimated the number of changes for these chromosomal traits, and found the highest for 5S loci. Patterns of character change are largely homoplastic, although combinations of traits can be useful to identify groups within Iochrominae. Asymmetry was the only character that allow us to differentiate this clade among its relatives. Overall, our study suggests that the diversification of Iochrominae has not been accompanied by the formation of strong chromosomal barriers, which may help to explain the crossability of many species and even genera within the group. Highlights • Iochrominae is a largely Andean clade known for its remarkable floral diversity • Ancestral karyotypes were reconstructed into a molecular phylogeny • Chromosomal changes are homoplastic, although combinations of traits identify groups within Iochrominae • Iochrominae comprises species with highly symmetrical karyotypes, mostly 2n = 24 • Diversification of Iochrominae has not been accompanied by strong chromosome barriers 2 Key Words asymmetry, chromosome, evolution, Iochrominae, karyotype, Solanaceae 1. Introduction The tomato family Solanaceae includes a diversity of economically important species, such as potatoes, chili peppers, eggplants and tobacco. All of these crops belong to a major lineage within the family informally called the "x = 12 clade" (Olmstead et al., 2008). Its roughly 2300 species share chromosome numbers based on 12 pairs, including the giant genus Solanum, with c. 1000-1500 species (Bohs, 2005). The conservation of this base chromosome number suggests that the diversification of this clade across all continents (except Antarctica) over roughly 20.921 million years ago (mya; Dupin et al., 2017;De-Silva et al., 2017) was not coupled with significant cytological evolution. Nonetheless, detailed cytological studies remain few, especially for diverse and poorly studied Neotropical groups. For example, there are only c. 52 chromosome counts for the entire tribe of tomatillo and its allies (Physalideae), comprising 29 genera and more than 200 spec...