Amazonian populations are characterized by establishing itinerant productive systems called chagras, where roots and tubers play an important role as energy sources due to their diversity. The aim of this research was to characterize the roots and tubers, apart from cassava, used by local communities in two locations of the Colombian Amazon: Vaupes and Amazonas. Both locations have different ethnical groups and soil origins belonging to tertiary and quaternary formations. The tuber and root diversity and related local use knowledge were assessed with non-structured interviews. The identity of the ethnovarieties was assessed by visiting chagras and stubble fields; the collected botanical samples were included in herbarium collections. Three ethnovarieties of roots and tubers were preliminarily characterized with phytochemical analyses, proximate composition, and primary metabolites quantification. Twenty-five ethnovarieties in Vaupes and eighteen in Amazonas (rhizomes, tuberous roots, corms and bulbils) were distributed in 7 families: Araceae, Marantaceae, Heliconiaceae, Zingiberaceae, Dioscoreaceae, Convolvulaceae and Cannaceae, with the first one being the most diverse. For the species Canna indica, Calathea allouia and Maranta ruiziana, the moisture content ranged from 63.9-87.5% and the proximate composition on a dry weight basis was below 9.6% proteins, 8.7% ash and 87.3% carbohydrates. The predominant organic acid was succinic and polysaccharides were the principal component (maximum of one third of the carbohydrate fraction corresponded to mono and disaccharides). Flavonoids, tannins and alkaloids were also detected. C. indica showed an antioxidant capacity. The characterization of the Amazonian ethnovarieties of roots and tubers will became a baseline for reinforcing the regional food sovereignty by developing new root and tuber based products.