A new species of Jenynsia from southern Brazil is described. Jenynsia weitzmani n. sp. from the rio Pinheiros drainage in Santa Catarina state is diagnosed by a single dark midlateral stripe, absence of associated dark dorsolateral or ventrolateral stripes or blotches in adults, a terminal to slightly oblique mouth, and absence of the first mandibular-canal pore (pore W). A phylogenetic analysis of primarily osteological data supported the new species as sister to a group composed of J. eirmostigma and J. eigenmanni within the subgenus Plesiojenynsia. A key to Jenynsia species is provided.