This work documents the biostratigraphy of dinoflagellate cysts from the La Barca Formation at the Punta Ainol Locality, Tierra del Fuego Province, Argentina. The succession at Punta Ainol represents a new record for the Paleogene (late Paleocene–middle Eocene) from southern South America, based on current data on nannofossils and dinoflagellate cysts. Some significant events are in agreement with nannofossil data. In ascending stratigraphic order, they include the first occurrences (FOs) of Impagidinium crassimuratum, Apectodinium homomorphum, Pyxidiniopsis waipawaensis, Samlandia septata, Cleistosphaeridium diversispinosum, and Enneadocysta dictyostila, and the latest occurrence (LO) of Palaeocystodinium golzowense. Three Paleocene/Eocene dinoflagellate cyst zones (Palaeocystodinium golzowense, NZDP7 and Apectodinium homomorphum) are recognized here.