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Integrated Water Resources Management (IWRM) is vital to the future of Malawi and motivates this study's provision of the first stable isotope baseline characterization of the Shire River Basin (SRB). The SRB drains much of Southern Malawi and receives the sole outflow of Lake Malawi whose catchment extends over much of Central and Northern Malawi (and Tanzania and Mozambique). Stable isotope (283) and hydrochemical (150) samples were collected in 2017–2018 and analysed at Malawi's recently commissioned National Isotopes Laboratory. Distinct surface water dry-season isotope enrichment and wet-season depletion are shown with minor retention of enriched signatures ascribed to Lake Malawi influences. Isotopic signatures corroborate that wet-season river flows mostly arise from local precipitation, with dry-season flows supported by increased groundwater contributions. Groundwater signatures follow a local meteoric water line of limited spread suggesting recharge by local precipitation predominantly during the peak months of the wet-season. Relatively few dry-season groundwater samples displayed evaporative enrichment, although isotopic seasonality was more pronounced in the lowlands compared to uplands ascribed to amplified climatic effects. These signatures serve as isotopic diagnostic tools that valuably informed a basin conceptual model build and, going forward, may inform key identified Malawian IWRM concerns. The isotopic baseline establishes a benchmark against which future influences from land use, climate change and water mixing often inherent to IWRM schemes may be forensically assessed. It thereby enables both source-water protection and achievement of Sustainable Development Goal 6. [ABSTRACT FROM AUTHOR]