Astyanax Baird & Girard, 1854 is the most widely distributed genus of Acestrorhamphidae, occurring from the West Texas Gulf to Central Argentina. Out of the 120 valid species currently recognized, around 15 occur in the Amazon basin. A possible new species was registered in streams of the Aripuanã River basin (= Madeira River basin) and Juruena River basin (= Tapajós River basin). For the new taxon, morphological, meristic, and coloration characters of 42 specimens were analyzed. Astyanax sp. n. differs from all its congeners by the following combination of characters: two humeral spots, the first vertically elongated and the second approximately rounded, beginning on the dark longitudinal stripe; maxilla toothless or with 1 tooth; premaxilla with two tooth rows: outer row with 4 or 5, tricuspids, inner row with 4 or 5, heptacuspid; dentary with 4 teeth, penta- to hexacuspid; complete lateral line, extending to the end of the caudal peduncle, with 34 to 37 perforated scales; 12 to 15 circumpeduncular scales; 6 or 7 transverse scale rows above the lateral line; 5 or 6 below; anal fin with 22 to 27 branched rays. Males of the new species show a straight anal fin and a bony hook on the distal segments of the last unbranched ray. The record of the new taxon highlights the importance of sampling headwaters of Amazonian drainages, which harbor significant undescribed species diversity, especially small-sized ones, in areas subject to anthropogenic impacts (e.g., extensive agriculture). The description of this new species expands the known diversity of Astyanax and contributes to reducing the Linnean shortfall of Neotropical ichthyofauna.