The chromosome numbers of 21 genera and 44 species of subfamily Epidendroideae belonging to tribes Sobralieae, Epidendreae, Malaxideae and Vandeae, and subtribe Dendrobiinae, were determined. Chromosome numbers varied from 2n = 24 in Malaxis pubescens to 2n = c. 240 in Epidendrum cinnabarinum. A revision of the chromosome numbers known for the subfamily was also performed, aimed at determining the basic numbers of the genera, subtribes and tribes. The first counts for 31 species and six genera of tribe Sobralieae and subtribe Ponerinae are presented. The basic number for each genus was evaluated. A predominance of x = 20 in genera of Epidendreae and Arethuseae, x = 19 in Vandeae and subtribe Dendrobiinae and x = 15 and 21 in Malaxideae was observed. Other tribes were more variable. A wide occurrence of x = 19 and 20 in Epidendroideae and of x = 21 in at least one genus of all tribes suggests that disploidy of one or a few chromosomes has played a decisive role in the establishment of the basic karyotypes. The karyotype variability observed in the subfamily is discussed in light of current phylogenetic proposals for the family. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 163, 234–278.