The aim of this research is to define a new Macrophyte Index relevant in the context of the Po Valley. The application of biotic indexes, as the macrophyte ones, is required by both the Italian and the European law to provide an adequate classification of freshwaters, in association with physical–chemical and hydromorphological data. At the same time, these methodologies, based on the sensibility of macrophytes towards pollution, could be an important mean to evaluate the quality of the waters coming from wetlands, providing information related to the depuration capabilities of these important ecotonal zones. In this study, we elaborate a floristic list of about 340 taxa; moreover, we chose 19 sampling sites, analysed physical–chemical parameters and obtained floristic data. These results, integrated by literature information on ecological needs, distribution and rarity of all species, allowed the attribution of indicator values. Thus, two new indexes have been elaborated: Indice Macrofitico di Pianura (I.M.P.), mostly accounting for water quality, and Indice di Biodiversità Riparia (I.B.R.), mostly accounting for biodiversity of macrophytes on the banks. The efficacy of the proposed indexes has been evaluated by comparing them to analysed physical–chemical parameters and other indexes used in Europe, such asMacrophyte Index Scheme (M.I.S), Indice Biologique Macrophytique en Rivière (I.B.M.R.) and Extended Biotic Index (I.B.E.).