Hemigrammus lunatus is distinguished from most congeners, except from H. barrigonae, H. machadoi and H. ulreyi, by having wide dark horizontal stripe across the eye (vs. eye stripes absent or, when present, vertical in the remaining congeners). It differs also from most congeners, with the exception of H. barrigonae, H. boesemani H. geisleri, H. machadoi, H. mimus, and H. ulreyi, by having well defined narrow dark stripe at the basis of the anal fin (vs. dark stripe at the basis of anal fin absent). It can be readily diagnosed from H. boesemani, H. geisleri, and H. mimus, by having a distinct roundish dark humeral blotch (vs. humeral blotch absent) and by lacking a blotch on caudal peduncle or any distinct patch of pigmentation on caudal fin (vs. blotch on caudal peduncle present in H. boesemaniand H. geisleri, a dark marking present at the basis of each caudal-fin lobe basis in H. mimus). It differs from H. barrigonae and H. ulreyi by lacking a narrow, well-defined longitudinal dark stripe (vs. present), and by lacking a discrete blotch on caudal peduncle (vs. present in H. barrigonae) or a dark pigmentation patch on the basis of anteriormost dorsal-fin rays (vs. present in H. ulreyi). It differs from H. machadoi by having the following characters: a small roundish dark humeral blotch, extending horizontally from fourth through sixth lateral-line scales, and vertically from fourth through fifth scale rows above lateral line (vs. conspicuous, vertically elongated humeral blotch, extending horizontally from second through sixth lateral-line scales, and vertically from third row above lateral line to first row below it); 6-7 gill-rakers on upper branch and 11-12 on lower (vs. 4-5, and 9-10, respectively); the lower number of cusps of inner premaxillary, dentary, and maxillary largest tooth (5 vs. 5-7 cusps, mode 7, in premaxillary and dentary; 1-3 vs. 3-5, mode 5, in maxillary); and 32-33 total vertebrae (vs. 34-35) (Ref. 96864).