<p><i>Mimosa</i> L. comprises approximately 620 species of socioeconomic and environmental importance, with about half occurring in the Brazilian Cerrado. Despite this diversity, cytogenetic studies in the genus remain limited, with most restricted to chromosome counts. We investigated 11 populations of seven <i>Mimosa</i> species, focusing on the heterochromatin distribution (CMA⁺ bands), and nuclear DNA content. Polysomaty was observed in root meristem cells of six species, absent only in <i>M. verrucosa</i>, in which only diploid cells were detected. Variation in the number, position, and intensity of CMA⁺ bands was detected both within and among populations. The number of CMA⁺ bands ranged from four in <i>M. caesalpiniifolia</i> (diploid cells) to 104 in <i>M. candollei</i> (octaploid cells), with no apparent phylogenetic pattern. Polysomatic cells generally showed a proportional increase in CMA⁺ bands with ploidy level. Nuclear DNA content varied from 1C = 0.64&#xa0;pg (<i>M. sensitiva</i>) to 1C = 1.55&#xa0;pg (<i>M. arenosa</i>), with no correlation (<i>r</i> = 0.1417) between ploidy and DNA content. Our results, together with previous reports, indicate that polysomaty is a frequent genomic feature in <i>Mimosa</i>, likely associated with environmental adaptation, growth and development. This study represents the first intra- and interpopulation assessment of heterochromatin variation and nuclear DNA content across multiple <i>Mimosa</i> species, providing novel insights into heterochromatin dynamics and genome organization in this diverse cytogenetically understudied legume genus. This article aligns with SDG 15 (Life on Land) of the UN Agenda for Sustainable Development.</p>