We present the first quantification of forest community composition and its relationship with environmental factors in South American subtropical Atlantic Forests. In this region, rain, seasonally dry and mixed forests form an ecotonal zone near the parallel of latitude 30°S. To investigate how well current knowledge on climatic effects and biogeographic distribution apply to subtropical ecotones, we tested the following expectations: (i) there is a floristic longitudinal gradient correlated to altitudinal and climatic gradients; (ii) climatic variables are more important than soil factors in shaping floristic composition; and (iii) there are three floristic regions in the southernmost limit of the Atlantic Forest biome that are expected to be distinct in composition, structure and biogeographical origin. We examined floristic composition and its relationship with environmental factors across 52 1-ha permanent study areas in subtropical Brazil, containing in total 269 tree species ≥ 9.5 dbh (diameter at breast height). Climatic data, related to rainfall seasonality and temperature, as well as soil properties, were compiled from published sources or global data banks. Expectations one and two were confirmed, but expectation three was only partially met. Hierarchical cluster analysis divided the southernmost Atlantic Forests into four major groups (Rain, Seasonally Dry, Western Mixed and Eastern Mixed Forests). Overall, the tested environmental variables differed significantly among the four regions. Using indicator species analysis, we distinguished 46 indicator species, which had significant environmental preferences for one floristic region. These species can be used as indicators of environmental conditions or to determine to which floristic region a certain forest belongs. Biogeographic distributions differed between floristic groups, supporting the interpretation that Eastern Mixed Forests are relict forests of a temperate forest of Andean origin that occurred during colder palaeoclimates. Western Mixed Forests represent the main floristic ecotone between Seasonally dry and Eastern Mixed Forests.