The Cordillera de los Andes is one of the most important regions for biodiversity. Among amphibians, many endemic species of terrestrial-breeding frogs have recently been discovered. Herein we describe Phrynopus sancristobali from the Andes of southeastern Peru based on molecular and morphological data. The new species is known from the ecotone between humid puna and montane forest at 3910 m a.s.l. on the left side of the Apurímac Valley in the Department of Ayacucho. The new species differs from congeners by having dorsum bearing pustules and light brown with dark brown reticulations surrounding the areolas, and coloration consisting of flanks golden brown with gray or dark brown marks, palms pale orange, soles deep orange, toes IV and V dark brown in dorsal and ventral view, belly cream to yellow with gray to light brown marks, and groin and throat deep orange. The snout-vent length (SVL) is 20.7 and 22.2 in two females, and 19.5 mm in one male. According to our phylogeny inferred using Maximum Likelihood with a concatenated dataset of three mitochondrial and two nuclear genes, P. sancristobali is sister taxon of P. apumantarum, recently described from Department Huancavelica. Our description extends the known geographic range of Phrynopus 73 km to the south, and P. sancristobali is the only species in the genus known to occur south of the Mantaro River, whose deep valley is hypothesized to be a biogeographic barrier for high-Andean organisms. The discovery of P. sancristobali confirms the high levels of endemism and beta diversity of Phrynopus in the moist puna grasslands and montane forests of the high Andes of Peru, and suggests that further work will reveal the presence of additional species in southern Peru.