Uncancylus Pilsbry, 1913, a neotropical genus of freshwater limpets, includes four species and the “variety” Ancylus concentricus bonariensis established by Strobel in 1874. Uncancylus concentricus (d'Orbigny, 1835) is the best-known of all species of the genus, both in terms of the morphology as well as its distribution. The goals of this study were to describe a new species of Uncancylus from São Paulo State, Brazil—Uncancylus paulistanus n. sp.—and raise the subspecies proposed by Strobel to the rank of species, providing a redescription of shell and morphology (muscle scars and radula). We also provide a molecular analysis that supports the anatomical results in comparison with other sequences of species of Ancylinae. The diagnostic characters of the new species include a shell with an apical angle of almost 90°; three small adhesive areas in the anterior region located between the muscular scars; and the rachidian tooth with five cusps and a long basal plate with height twice its base. Uncancylus bonariensis (Strobel, 1874) has a shell with apical angle of 45°; three small adhesive areas located in the posterior region of the body; the rachidian tooth with five cusps and a basal plate with height twice its longer base. Molecular analyses (both ML and BI) showed a dichotomy separating the genera of the Holarctic region from the Neotropical region. Also, both analyses were congruent showing similar topologies with high support values for the clade formed by Uncancylus concentricus, U. paulistanus n. sp., and U. bonariensis indicating that they are different species.