A new species of the Hypsiboas polytaenius clade is described from Itapecerica da Serra (23°43′S, 46°51′W, 906 m elevation; datum WGS84), State of São Paulo, Southeastern Brazil. The new species is characterized by its medium snout-vent length (SVL) for the group (SVL 27.1–29.1 mm in males; 31.2 mm in female), head width 31.1–33.9% of SVL in males, 37.8% in female, snout rounded in dorsal and lateral views, calcar appendix and supra-cloacal crest absent, webbing poorly developed between fingers, moderately developed between toes, and dorsal color pattern composed of four wide pale brown stripes, intercalated by three brown stripes, arranged longitudinally. The four pale brown stripes join to form two stripes anterior to the eyes and then join again at the tip of snout. Within these pale brown stripes, narrow longitudinal brown lines, complete or broken, occur. The pale brown marginal stripes show, on the external border, a white line passing over the canthus rostralis, edge of the upper eyelid, and along the flank to the groin, contrasting with a broad dark brown lateral stripe. This dark brown stripe extends from the tip of snout to the eye and from the posterior border of the eye, passing on the tympanum, to the groin. The ventral half of the loreal region is pale brown with a dark brown longitudinal line, continuing as a white stripe below and contrasting to the dark brown lateral stripe, to the groin. The new species is distributed in the extreme south of the State of Rio de Janeiro and northeastern State of São Paulo, above 400 m altitude.