In this study, the diffusive gradient in thin film (DGT) has been developed using three new materials derived from benzoylthiourea (PBTU, BTP1 and BTP2) for the determination of bioavailable mercury (Hg). The efficiency of these DGT devices was compared with the well-established 3-mercaptopropyl functionalized silica gel (3MFS) in laboratory and field conditions. It was found that PBTU lacked of consistency for handling and assembling in the DGT device, whereas the other two polymers provided mechanically stable binding gels and extracted satisfactorily Hg. However, BTP2 showed no correlation between Hg uptake and Hg present in the aqueous solution, and therefore, BTP1 gels was chosen as the binding layer used for DGT technique. The calculated diffusion coefficient of DGT-BTP1 was 4.0 × 10 −6 cm 2 s −1 at 25 °C in 0.8 mm thick diffusion layer and 2.3% w/v of material in gel, whereas for DGT-3MFS, the D value was 9.1 × 10 −6 cm 2 s −1 . Moreover, it was found that neither the pH (range 4.5 – 9.5) nor the ionic strength (range 0.005–0.1 mol L −1 ) significantly affected the binding behavior of Hg (p > 0.05). Finally, the DGT-BTP1 devices were successfully used for in-situ measurements of Hg in Quito River (Colombia), impacted by artisanal gold mining, and were compared with DGT-3MFS. Results showed a good performance with values of the labile Hg concentration between 5 to 8% of the total Hg in water. This study demonstrates that benzoylthiourea based DGT is a useful tool for in situ monitoring of Hg in freshwaters. • Three new materials derived from benzoylthiourea were used for DGT technique. • BTP1 extracted satisfactorily Hg, with a diffusion coefficient of 4.0 × 10 −6 cm 2 s −1 . • DGT-BTP1 was suitable for aqueous solutions with a wide pH and ionic strength range. • These devices were successfully used in a Colombian river highly impacted by Hg. • Benzoylthiourea based DGT was a useful tool for in situ monitoring of Hg in freshwaters.