Industrial expansion in the North Bohemian Coal District over the past decades has had an adverse effect not only on stream sediments but also on the freshwater system. So far mercury occurrence has not been properly investigated in the area. In response to this situation, the present study applies mercury quantification and speciation in order to predict the possible mobility of mercury. The enrichment factor (EF) and sediment quality guidelines (SQGs) were used to calculate the degree of mercury contamination. An automatic mercury analyser was employed to detect the total mercury content (Hg-T) and Thermal Desorption technique was used to identify mercury species. All the sediment samples were assessed with the contamination degree which increased to very severe in stream sediments in the middle section of the river course. Mercury species identified were chiefly the insoluble forms Hg-FeS2 and HgS. Lignite components and combustion residues were identified as the primary source of Hg contamination in the area.