The well-exposed marine Eocene units from southwestern Patagonia, Argentina, contain useful information for reconstructing regional climate and oceanographic patterns in an area adjacent to the Drake Passage. The aim of this paper is to integrate dinoflagellate cyst data from three sections of the southwestern Austral Basin (Río Turbio Formation) to propose a zonation scheme, which can be applied to other southwestern Atlantic Ocean sites. Assemblages of organic walled dinoflagellate cysts have been analysed in different cropping-out sections and cores, showing the high potential of this fossil group as biostratigraphic markers. Comparison of dinoflagellate cyst events of the upper member of the Río Turbio Formation with calibrated biostratigraphic ranges in the Palaeogene South Pacific Ocean allowed us to date and correlate these sedimentary sections. The resulting zonation consists of four dinoflagellate cyst zones labelled RTF 1 to RTF 4, between the middle Lutetian and late Priabonian. As a final point, we applied dinoflagellate cyst species with importance as palaeoenvironmental markers to assess long-term climatic and oceanographic evolution for the area. This study shows that the endemic–Antarctic dinoflagellate cyst assemblage is dominant during the middle to late Eocene (RTF 1 to RTF 3), while a significant replacement of these taxa by cosmopolitan species characterizes the upper part of the upper member of the Río Turbio Formation (RTF 4). This turnover seems to be a consequence of changes in the ocean circulation patterns forced by deepening of the southern Atlantic gateways (the Drake Passage and the Tasman Gateway).