Besleria naquenensis is described and illustrated on the basis of a collection from the understory of seasonally flooded forests drained by black waters located in the lower “Río (Caño) Naquén,” northwestern base of “Serranía de Naquén,” upper Guainía River basin, Guainía Department, Colombia, and its relationships with related species are discussed. This new species shares features with three species (B. gibbosa, B. neblinae, and B. yatuana) of section Neobesleria subsection Axillares. Morphologically, it is closest to B. yatuana, but it differs in its leaves, inflorescences, calyx, and corolla. In addition, habitat information, a distribution map, and data of its conservation status are included. An identification key of 27 species of Besleria found in the Rio Negro basin (Brazil, Colombia, and Venezuela) and adjacent regions is also provided. This new species elevates to 66 the number of Besleria taxa for the flora of Colombia, the country with the highest diversity of the genus.