We submitted tree species occurrence and geoclimatic data from 59 sites in a river basin in the Atlantic Forest of southeastern Brazil to ordination, ANOVA, and cluster analyses with the goals of investigating the causes of phytogeographic patterns and determining whether the six recognized subregions represent distinct floristic units. We found that both climate and space were significantly (p ≤ 0.05) important in the explanation of phytogeographic patterns. Floristic variations follow thermal gradients linked to elevation in both coastal and inland subregions. A gradient of precipitation seasonality was found to be related to floristic variation up to 100 km inland from the ocean. The temperature of the warmest quarter and the precipitation during the coldest quarter were the main predictors. The subregions Sandy Coastal Plain, Coastal Lowland, Coastal Highland, and Central Depression were recognized as distinct floristic units. Significant differences were not found between the Inland Highland and the Espinhaço Range, indicating that these subregions should compose a single floristic unit encompassing all interior highlands. Because of their ecological peculiarities, the ferric outcrops within the Espinhaço Range may constitute a special unit. The floristic units proposed here will provide important information for wiser conservation planning in the Atlantic Forest hotspot.