The Epidendrum nocturnum Jacq. group comprises about 60 species. Nine species occur in Brazil, predominantly in the Atlantic and Amazon Forests. The group is taxonomically complex because its species are morphologically similar and not easily distinguished. The main objective of this work is to characterize their chromosome evolution and how this has affected species delimitation in Brazilian representatives of the E. nocturnum group. We used chromosome numbers, heterochromatin band patterns, and genome size variation to better understand the chromosome variation, species delimitation, and the relationship among seven representatives of this group. A new species from the Cerrado/Amazon Rainforest ecotone is described based on cytological and morphological characters. The new species, Epidendrum pareciense sp. nov., is a diploid (2n = 40), Epidendrum bahiense Rchb.f., Epidendrum carpophorum Barb.Rodr., Epidendrum micronocturnum Carnevali & G.A.Romero, Epidendrum purpureocaulis Sambin & Essers, and E. nocturnum are tetraploids (2n = 80), and Epidendrum tumuc-humaciense (Veyret) Carnevali & G.A.Romero is hexaploid (2n = 120), the last a new ploidy for the group. Heterochromatin in these species is characterized by guanine-cytosine-rich regions (cromomycin A3 (CMA+) bands) on proximal and terminal regions associated with NORs. Genome size and numbers of CMA terminal bands are directly related to ploidy, suggesting that diploidization has not yet occurred and supporting the hypothesis of a recent polyploid origin for the E. nocturnum group.