Karyotype can provide information about taxonomic relationships, genetic aberrations and the evolutionary origins of species. However, differentiation of the tiny chromosomes of Brachiaria mutica and creation of a standard karyotype for this grass has not been accomplished due to lack of distinguishing features and polyploidy. In this study, somatic chromosome counts determined in 102 individuals from 34 B. mutica populations and selected for karyotype analysis. Among them the diploid plants have 2n = 36 = 5M + 9 m + 4sm karyotype. In combination with previous chromosome data, the present study reveals a uniform basic chromosomal number (2n = 36) and uniformity of karyotype indicating that speciation through polyploidization is less likely in B. mutica, despite the highly diversified habitats it occupies.