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The tradition of floristic studies in Italy has made it possible to obtain a good knowledge of plant diversity both on a national and regional scale. However, the lack of knowledge for some areas, advances in plant systematics and human activities related to globalization, highlight the need for further studies aimed at improving floristic knowledge. In this paper, based on fieldwork and herbaria and literature surveys, we update the knowledge on the Italian vascular flora and analyze the floristic similarities between the administrative regions. Four taxa, all exotic, were recorded for the first time in Italy and Europe. In detail, Elaeodendron croceum, Kalanchoë blossfeldiana, and Sedum spathulifolium var. spathulifolium were found as casual aliens, while Oxalis brasiliensis was reported as historical record based on some herbarium specimens. Furthermore, Kalanchoë laxiflora was confirmed as a casual alien species for Italy and Europe. Status changes for some taxa were proposed at both national and regional levels, as well as many taxa were reported as new or confirmed at the regional level. Currently the Italian vascular flora comprises 9150 taxa of which 7547 are native (of which 1598 are Italian endemics) and 1603 are exotic at the national level. The multivariate analysis of updated floristic data on a regional scale showed a clear distribution along the latitudinal gradient, in accordance with the natural geographical location of the regions in Italy. This pattern of plants distribution was not affected by the introduction of alien species. Despite some taxonomic and methodological issues which are still open, the data obtained confirm the important role of floristic investigations in the field and in herbaria, as well as the collaborative approach among botanists, in order to improve the knowledge of the Italian and European vascular flora.