This study was undertaken to assess the impact of combined sewer overflows (CSOs) on distribution and potential mobility of heavy metals in sediments of urban streams in Prague, Czech Republic. Contents of total and extractable heavy metals (Cu, Zn, Pb, Cd, Cr and Ni), mineralogical phases and other sediment properties were measured in 44 surficial sediment samples. Total metal concentrations were obtained after microwave-assisted digestion whilst extractable metal contents were obtained following a sequential extraction scheme (acid soluble, reducible, oxidisable and residual fraction). The multivariate statistics of cluster analysis was used to identify specific areas of contamination and to evaluate the impact of CSOs. The observed mobility order of metals was Cd > Zn > Ni > Cu > Pb > Cr. There was a considerable increase in Zn mobility and increase of Cu associated with the oxidisable fraction in the sediments below CSO discharges. Cd was revealed as the most mobile heavy metal with percentages of extraction of approximately 40-60% in acid soluble fraction. Pb was mainly found in reducible fraction associated with Fe/Mn (oxi)hydroxides, which is indicative of anthropogenic pollution. In terms of environmental significance, Cd and Zn can be particularly mobile and bioavailable under acidic conditions, because they are predominantly bound in labile fractions. However, potential changes of redox state and pH may remobilize the metals bound to carbonates, reducible, and/or organic matter.