A new Habenaria species from the state of Mato Grosso is described and illustrated. Habenaria gracilisegmenta was discovered in campinarana understory, in northern Mato Grosso state, on the southern edge of the Brazilian Amazon. The species is distinguished by its slender habit, and by the few, delicate flowers, with very long, thin lateral petals and lip segments. Its morphological affinity with other Neotropical species is unclear. A molecular phylogenetic analysis revealed that the new species does not belong to any of the Neotropical subclades of the genus, constituting an additional lineage. The species is one of two of the genus endemic to the Brazilian Amazon, and considered threatened given the small number of known populations and restricted distribution.