Abstract The lower sector of the Guadiana River Basin, located in southern Portugal and Spain, hosts soils and plants with elevated Pb, Cu and As near former and current mining sites. Two geogenic and two anthropogenic sources of Pb were identified where elevated concentrations are mostly related to the occurrence of sulphide-rich ore deposits, Volcanic Sedimentary formations and mining. These were generally reflected by the multiple regression analysis (MRA) and confirmed by isotope determinations. Nevertheless, caution was needed in interpreting statistical and isotopic results; therefore the combination of both techniques was important. Elements, such as Ca, Na, Cu and As, show enrichment in soil developed on shale, sandstone and conglomerate of Upper Devonian age belonging to the Phyllite-Quartzite Group. Lead exhibits an enrichment in soil developed on felsic volcanic rocks from the Volcanic Sedimentary Complex (VSC) of Upper Devonian-Lower Carboniferous age, which has been identified by the relationship between topsoil median values of different lithologies and grand subsoil median values. In the same soil, Fe, As, Co, Ni and Cr are depleted. Translocation of Pb to the aerial parts of plants is insignificant in all three plant species studied and analysed ( Cistus ladanifer L., Thymus vulgaris , Lavandula luisieri ). High Pb concentrations in soil, where Cistus ladanifer L. developed, the only representative number of species analysed, do not correspond generally to elevated Pb contents in plants, except near mine sites, where lower pH of soil, increases Pb bioavailability. The different statistical methodologies combined with Pb isotopic studies were successfully applied in the identification of Pb sources in soil and Cistus ladanifer L. plant of the Lower sector of Guadiana River basin. Therefore, rocks, mineralisations, subsoil, topsoil and plant processes were successfully integrated to understand the migration of Pb into the food chain.