Southern Hemisphere fossil plants indicate a Late Cretaceous and Early Paleogene rainforest biome across much of Gondwana, but global cooling and separation of South America and Australia from Antarctica are linked to significant ecological change during the middle and late Eocene. The Río Pichileufú fossil flora (RP) from Río Negro Province, Argentina offers an exceptional opportunity to observe the earliest stages of floral response to cooling in Patagonia and biogeographic signals of South American isolation. The principal record of Patagonian fossil plants during the Early Eocene Climatic Optimum (EECO) is from Laguna del Hunco (LH) in Chubut Province, Argentina, with an ⁴⁰Ar/³⁹Ar age of 52.22 (±0.22) Ma. RP has an ⁴⁰Ar/³⁹Ar age of 47.74 ±0.05 Ma, ca. 3 Ma after the EECO, during cooling and separation of South America from Antarctica. Previous work indicates that although many plant taxa are found at both sites, including the conifers Dacrycarpus and Agathis, RP lacks taxa observed at LH, including the angiosperms Eucalyptus and Gymnostoma. Additionally, taxa previously observed at RP are not represented at LH, including Asteraceae flowers. The 1938 E.W. Berry RP Type and Cohort collection, still represents the most diverse described Cenozoic fossil flora from South America. However, Berry’s collection provides only a qualitative record of RP, and just a fraction of its specimens have received modern paleobotanical treatment. To prepare for quantitative tests of paleofloral response to cooling and biogeographic change, I begin with a comprehensive update of RP, including consideration of more than 1100 specimens from recent, unbiased RP collections. A project database, developed using FileMaker Pro Advanced v. 14.0.4, provides data management and automated leaf architecture descriptions to assist manuscript preparation for this work. Here, I present new evidence from the combined RP collections, supported by morphotype characterization based on the Manual of Leaf Architecture, 2009. Through future quantitative comparisons of the RP and LH assemblages, I predict that shifts in floral relative abundance will be observed as well as net loss of ancient Gondwanan rainforest associations and net gain of new occurrences.