Morphological boundaries between South American species of Euphrasia L. are controversial, rendering determination of specimens an arduous task. In this context, a comprehensive taxonomic revision of Euphrasia in South America is here provided for the first time. This study, based upon a classical morphological study of ca. 400 herbarium specimens, supports the recognition of eight species and one subspecies distributed in the Andean regions of Argentina, Bolivia, and Chile. From among native species, six belong to section Trifidae Benth. and one to the monotypic section Paradoxae Pugsley, endemic to Juan Fernández Islands; one adventive species, E. officinalis L., belongs to the section Euphrasia. The previously misunderstood presence of E. cockayniana Petrie is here untangled, and, consequently, the species is excluded from South America. A key to all Euphrasia taxa in South America, plus morphological descriptions, nomenclature items, geographical distribution and maps, habitat notes, illustrations, photographs, and discussion notes are included for the nine taxa. Eleven names are here synonymized, and lectotypes are designated for E. andicola Benth., E. debilis Wettst., E. flavicans Phil., E. intricata Phil., and E. philippii Wettst. Euphrasia andicola is reported for the first time for Argentina. This collaborative effort will represent a baseline for further investigations on Euphrasia in South America.