The macroscopic algae Characeae are usually assumed to occur in waterbodies supplied by groundwater with low phosphate content, but the indicative value of the species is seldom defined in bibliography. Former braided channels of the Rhône river are supplied with groundwater originating from the main channel (seepage) or from hillslope aquifer. The aim of the present paper was to determine if it possible to use the Characeae as indicators of physicochemical characteristies of water in order to assess the origin of groundwater supplying former river channels. Four former braided channels of the Rhône River colonized by Characeae were investigated, and the physico-chemical characteristics of i) the channels, ii) the groundwater and iii) the river were measured over a period of several months. Species are arranged along a gradient of conductivity, alkalinity, ammonium and phosphate content of the water. Charophyte species can indicate the origin of groundwater, either seepage or hillslope nutrient-poor aquifer, and integrate both the average value of the chemical parameter, and their variations. C. hispida occurs in a nutrient-poor channel mainly supplied by highly calcareous groundwater coming from hillslope aquifer. Chara major has requirements close to those of C. hispida, but is more tolerant to periodic inputs of nutrients. C. vulgaris and N. syncarpa both tolerate mesotrophic waters originating from both hillslope aquifer and seepage, and C. globularis is associated to a channel mainly supplied by mesotrophic to eutrophic river seepage.