We analyzed and described quantitative differences in morphology and calls of Physalaemus petersi and P. freibergi, both members of the monophyletic Physalaemus pustulosus species group. We found significant differences between the two species in both morphometric and call parameters. Physalaemus petersi has proportionately longer legs and a narrower dorsum and head than P. freibergi. The calls of P. petersi are higher in frequency and longer than P. freibergi. Discriminant Function Analysis (DFA) of morphometric variables correctly classified 76.7–87.4% of individuals to species. DFA of call variables correctly classified 96.8–100.0% of males to species. Physalaemus petersi is found north of the Río Marañon and Río Amazonas in eastern Ecuador, northeastern Peru, and southeastern Colombia; P. freibergi is found south of these rivers in Amazonian Brazil, southeastern Peru, and Amazonian Bolivia. Calls and geographic locations are the most reliable means of identifying these species in the field.