In this study, we analysed chromosome number variation and chromomycin A3/4′,6‐diamidino‐2‐phenylindole (CMA/DAPI) banding patterns in 48 species belonging to 12 genera of subtribe Pleurothallidinae (Orchidaceae) in order to understand the chromosome evolution based on recent phylogenetic hypotheses and taxonomic treatments. All species had small chromosomes, with numbers ranging from 2n = 20 in two Specklinia spp. to 2n = 80 in an unidentified Octomeria sp. In Acianthera, the most highly represented genus in this study, a great diversity of chromosome number and pattern of fluorescent bands was observed, showing heterochromatin accumulation in Acianthera section Sicariae subsection Pectinatae. Interspecific ascending and, mainly, descending dysploidy were the main mechanisms of chromosome number evolution in subtribe Pleurothallidinae. For Pleurothallidinae, x = 20 is suggested as the basic chromosome number, the same suggested for the related subtribe Laeliinae and for the whole tribe Epidendreae. The Brazilian species of the mega‐genus Stelis had chromosomes with small amounts of heterochromatin and chromosome numbers based on x2 = 16. These are generally divergent from those reported for Andean and Meso‐American species, but in agreement with the monophyletic hypothesis proposed for Stelis spp. with a Brazilian Atlantic distribution. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178, 102–120.