Sediments of the Elbe River have been extremely polluted by contaminants originating from previous large-scale hexachlorocyclohexane (HCH) production and the application of γ-HCH (lindane) in its catchment in the second half of the twentieth century. In order to gain knowledge on bioaccumulation processes at lower trophic levels, field investigations of HCHs in macroinvertebrates were carried out along the longitudinal profile of the Elbe and tributary. Among the sites studied, concentrations in macroinvertebrates ranged within five orders of magnitude (0.01–100 μg/kg). In general, lower values of HCH isomers were observed at all Czech sites (mostly <1 μg/kg) compared with those in Germany. At the most contaminated site, Spittelwasser brook (a tributary of the Mulde), extremely high concentrations were measured (up to 234 μg/kg α-HCH and 587 μg/kg β-HCH in Hydropsychidae). In contrast, the Obříství site, though also influenced by HCH production facilities, showed only negligibly elevated values (mostly <1 μg/kg). Results showed that fairly high levels of α-HCH and β-HCH compared to γ-HCH can still be detected in aquatic environments of the Elbe catchment, and these concentrations are decreasing over time to a lesser extent than γ-HCH. Higher HCH concentrations in sediments in the springtime are considered to be the result of erosion and transport processes during and after spring floods, and lower concentrations at sites downstream are thought to be caused by the time lapse involved in the transportation of contaminated particles from upstream. In addition, comparison with fish (bream) data from the literature revealed no increase in tissue concentrations between invertebrates and fish.