Hypochaeris is thought to have arrived in South America by dispersal over the Atlantic Ocean from NW Africa during the Pliocene or Pleistocene. We used amplified fragment length polymorphism (AFLP) to unravel specific limits and relationships in the South American group of Hypochaeris (c. 45 species). The Moroccan endemic Hypochaeris angustifolia, which is sister to the entire South American group, was used as outgroup. Our AFLP analysis comprises 415 individuals from 32 South American species and is based on six primer combinations with 670 fragments scored. It provides important information for the delimitation of species and detection of closely related species pairs or groups. Most species are monophyletic and supported with > 90% bootstrap proportion. Hybridization is suggested between Hypochaeris chillensis and H. microcephala in Brazil. The internal nodes (or “backbone”) of the tree are not highly supported, but six major phylogenetic groups (also showing similarity in distribution and growth form) center around Hypochaeris apargioides, H. chondrilloides, H. microcephala, H. pampasica, H. sessiliflora, and H. tenuifolia. These results suggest that rapid migration into different geographical regions played an important role in the initial diversification of Hypochaeris in South America.