The adverse impacts of detrimental thallium (Tl) contamination are of increasing concerns to sustainable development. Herein, the contents, distributions and sources of Tl and potential toxic elements (PTEs) (Pb, As, Cr, Cu, Ni, Co, Sb, Cd and U) were investigated in sediments collected in Gaofeng River, which has been contaminated by long-term mining activities, located in Yunfu City, Southern China. Results indicated that excessive Tl levels were found in sediments (1.80–16.70 mg/kg). Sequential extraction procedure indicated that despite a large amount of Tl entrapped in residual fraction, a significant level of Tl (0.28–2.34 mg/kg) still exhibited in geochemically labile fractions, which was easy for Tl mobilization and availability. Pb isotope tracing method further confirmed that the pyrite exploitations may be the prime contaminated contributor (47–76%) to these sediments. These findings highlight that it is essential to establish more effective measures for Tl contamination control and call for engineered remediation countermeasures towards polluted river sediments.