A new species of Moenkhausia is described from tributaries of the upper rio Sepotuba, Paraguay basin, Brazil. The new species is distinguished from its congeners by a combination of characters, including an inconspicuous oval-shaped vertically elongated humeral blotch, extending horizontally from third through five lateral-line scales, and vertically from third row above lateral line to first row below it, followed by a diffuse field of dark chromatophores in the flank, combined with a well-defined dark line at the base of the anal fin. Furthermore, the phylogenetic position of the new species is presented based on molecular data, showing a close relationship among species of Moenkhausia and Hemigrammus that have a well-defined dark line at the base of the anal fin. Until this moment, this species is only known from in the upper rio Sepotuba basin.