Small shallow lakes (SSL) support exceptionally high and original biodiversity, providing numerous ecosystem services. Their small size makes them especially sensitive to anthropic activities, which cause a shift to dysfunctional turbid states and induce loss of services and biodiversity. In this study we investigated the relationships between environmental factors and macrophyte communities. Macrophytes play a crucial role in maintaining functional clear states. Better understanding the factors determining the composition and richness of aquatic plant communities in least-impacted conditions may be useful to protect these shallow lakes. We inventoried macrophyte communities and collected chemical, climatic, and morphological data from 89 least-impacted SSL widely distributed in France. SSL were sampled across four climatic ecoregions, various geologies, and elevations. Hierarchical cluster analysis showed a clear separation of four macrophyte assemblages strongly associated with mineralization. Determinant factors identified by distance-based redundancy analysis (db-RDA) analysis were, in order of importance, geology, distance from source (DIS, a proxy for connectivity with river hydrosystems), surface area, climate, and hydroperiod (water permanency). Surprisingly, at a country-wide scale, climate and hydroperiod filter macrophyte composition weakly. Geology and DIS are the major determinants of community composition, whereas surface area determines floristic richness. DIS was identified as a determinant in freshwater lentic ecosystems for the first time.