A first investigative screening of a wide-range chemical pollutants in surface water and biota samples has been conducted within the EUWI+ project in October 2020 at 27 sites in the Dnieper River Basin. Novel methodologies of wide-scope target screening of >2,200 and suspect screening of >65,000 or-ganic pollutants were used for analysis of each sample. The results were complemented with target analyses of metals. Legacy pollutants brominated diphenylethers (BDEs) and dioxins and dioxin-like compounds were analysed in biota samples. The results provided a first overview on the pattern and extent of chemical pollution in the Dnieper River Basin, where several pollution ‘hot-spots’ were identi-fied. Consequently, a follow-up investigation aiming at pollution cessation was recommended.