A new Hemigrammusis described from the rio Paraguai and rio Madeira basins, Mato Grosso and Rondônia States, Brazil. The new species is characterized by possessing a wide dark horizontal stripe across the eye, a vertically elongated humeral blotch, and 4-5 gill-rakers on upper branch and 9-10 on lower. The new species can be easily diagnosed from H. lunatus, the sympatric and morphologically most similar congener, by the shape of humeral blotch and the number of gill rakers. Data of the type material of both Hemigrammus lunatus and H. maxillaris, as well as extensive examination of specimens, allowed us to conclude that H. maxillarisis a junior subjective synonym of H. lunatus. A redescription of H. lunatus, as well as a formal restriction of its type locality, is provided. A putative monophyletic group within Hemigrammus, composed by H. barrigonae, Hemigrammus lunatus, H. machadoi new species, and H. ulreyi, named Hemigrammus lunatus group, is proposed based on overall body morphology and color pattern. Additionally, a discussion on the biogeographical relationships between the rio Paraguai and rio Guaporé basins is provided.