Hemigrammus xaveriellus sp. nov. is described from the upper Río Vaupés basin (Amazon basin), Departamento Guaviare, Colombia. It is distinguished from all congeners by the combination of the following characters: presence of a conspicuous, dark, longitudinal midlateral stripe extending along the body; presence of a conspicuous rounded, horizontally elongated humeral blotch anterior to the beginning of the dark midlateral stripe; seven scale rows between the dorsal fin and lateral line (vs. five to six); and five scale rows between the lateral line and pelvic‐fin insertion (vs. three to four). The single mature male of He. xaveriellus possessed a well‐developed urogenital papilla, an unusual feature among characids. The presence of an enlarged urogenital papilla in the family is discussed, and comments regarding the putative relationships of the new species are presented.