A comprehensive checklist of the Tuscan woody flora providing sub-regional details is presented. This checklist comprises 412 taxa, of which 264 are native and 148 are non-native. Nine taxa new for the region were found, and eleven taxa were excluded from the regional flora. The native taxa belong to 109 genera and 53 families. Rosaceae is the family richest in taxa, with Rosa being the genus with the highest number of species. On the provincial level, the province of Grosseto is the province with the highest number of native taxa, followed by the provinces of Lucca and Florence. The lowest number of taxa was documented in the provinces of Pistoia and Prato. With regard to the non-native woody flora, the highest number of non-native taxa can be found in the province of Livorno, whereas the lowest number occurs in the provinces of Arezzo and Siena. At regional level, 9% of non-native taxa are invasive, 45% naturalized and 46% casual. Asia and North America are the most common native ranges of those taxa. After an extensive herbarium study, the taxonomy and distribution of Acer opalus, Cornus sanguinea, Juniperus deltoides, Oreoherzogia alpina, Pyrus communis, Quercus crenata, Salix elaeagnos, and Sorbus aria are discussed in more detail.